*Article* **GC-MS Analysis of Biological Nitrate and Nitrite Using Pentafluorobenzyl Bromide in Aqueous Acetone: A Dual Role of Carbonate/Bicarbonate as an Enhancer and Inhibitor of Derivatization**

**Dimitrios Tsikas**

**Citation:** Tsikas, D. GC-MS Analysis of Biological Nitrate and Nitrite Using Pentafluorobenzyl Bromide in Aqueous Acetone: A Dual Role of Carbonate/Bicarbonate as an Enhancer and Inhibitor of Derivatization. *Molecules* **2021**, *26*, 7003. https://doi.org/10.3390/ molecules26227003

Academic Editor: Paraskevas D. Tzanavaras

Received: 1 November 2021 Accepted: 18 November 2021 Published: 19 November 2021

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Core Unit Proteomics, Institute of Toxicology, Hannover Medical School, 30625 Hannover, Germany; Tsikas.dimitros@mh-hannover.de

**Abstract:** Carbon dioxide (CO<sup>2</sup> ) and carbonates, which are widely distributed in nature, are constituents of inorganic and organic matter and are essential in vegetable and animal organisms. CO<sup>2</sup> is the principal greenhouse gas in the atmosphere. In human blood, CO2/HCO<sup>3</sup> <sup>−</sup> is an important buffering system. Inorganic nitrate (ONO<sup>2</sup> <sup>−</sup>) and nitrite (ONO−) are major metabolites and abundant reservoirs of nitric oxide (NO), an endogenous multifunctional signaling molecule. Carbonic anhydrase (CA) is involved in the reabsorption of nitrite and nitrate from the primary urine. The measurement of nitrate and nitrite in biological samples is of particular importance. The derivatization of nitrate and nitrite in biological samples alongside their <sup>15</sup>N-labeled analogs, which serve as internal standards, is a prerequisite for their analysis by gas chromatography–mass spectrometry (GC-MS). A suitable derivatization reagent is pentafluorobenzyl bromide (PFB-Br). Nitrate and nitrite are converted in aqueous acetone to PFB-ONO<sup>2</sup> and PFB-NO<sup>2</sup> , respectively. PFB-Br is also useful for the GC-MS analysis of carbonate/bicarbonate. This is of particular importance in conditions of pharmacological CA inhibition, for instance by acetazolamide, which is accompanied by elevated concomitant excretion of nitrate, nitrite and bicarbonate, as well as by urine alkalization. We performed a series of experiments with exogenous bicarbonate (NaHCO<sup>3</sup> ) added to human urine samples (range, 0 to 100 mM), as well as with endogenous bicarbonate resulting from the inhibition of CA activity in healthy subjects before and after ingestion of pharmacological acetazolamide. Our results indicate that bicarbonate enhances the derivatization of nitrate with PFB-Br. In contrast, bicarbonate decreases the derivatization of nitrite with PFB-Br. Bicarbonate is not a catalyst, but it enhances PFB-ONO<sup>2</sup> formation and inhibits PFB-NO<sup>2</sup> formation in a concentration-dependent manner. The effects of bicarbonate are likely to result from its reaction with PFB-Br to generate PFB-OCOOH. Nitrate reacts with concomitantly produced PFB-OCOOH to form PFB-ONO<sup>2</sup> in addition to the direct reaction of nitrate with PFB-Br. By contrast, nitrite does not react with PFB-OCOOH to form PFB-NO<sup>2</sup> . Sample acidification by small volumes of 20 wt.% aqueous acetic acid abolishes the effects of exogenous and endogenous bicarbonate on nitrite measurement.

**Keywords:** acetazolamide; carbonic anhydrase; derivatization; enhancement; GC-MS; inhibition; pentafluorobenzyl bromide

#### **1. Introduction**

Pentafluorobenzyl bromide (2,3,4,5,6-pentafluorobenzyl bromide, PFB-Br) is a useful derivatization reagent for different classes of organic substances including carboxylic acids and amines, as well as inorganic anions, including nitrate and nitrite [1]. Derivatization with PFB-Br can be performed in water-free organic solvents such as acetonitrile, as well as in aqueous systems in the presence of water-miscible organic solvents such as acetone. Use of an acetone-aqueous sample in a volume ratio of 4:1 enables derivatization in homogenous phase [1]. Reactions with PFB-Br are nucleophilic substitutions of bromide by

a nucleophile, which can be water, halogenides such as chloride, and other inorganic ions. The derivatization time is dependent upon the nucleophilicity and other factors, such as the stability of the PFB derivatives. The reaction products are lipophilic and extractable into organic solvents such as toluene and are best suitable for gas chromatography–mass spectrometry (GC-MS) analysis. Due to the fluorine atoms in PFB derivatives, their GC-MS analysis in the negative-ion chemical ionization mode revealed the highest sensitivity.

We previously developed a GC-MS method for the simultaneous quantitative measurement of nitrite and nitrate in different biological samples including human plasma, urine and saliva [2]. The reaction of PFB-Br with nitrite in aqueous acetone leads to the formation of the nitro derivative (PFB-NO2), yet not of the expected nitrous acid ester (PFB-ONO) (Scheme 1, reaction A). The reaction of PFB-Br with nitrate generates the nitric acid ester (PFB-ONO2) (Scheme 1, reaction B). Kinetic investigations showed that nitrite reacts with PFB-Br more rapidly and to a higher extent than nitrate, even at room temperature [2]. Yet, PFB-NO<sup>2</sup> seems to be readily susceptible to hydrolysis. As a compromise, we measured nitrite and nitrate simultaneously using GC-MS after derivatization with PFB-Br at 50 ◦C for 60 min. This procedure enhances the yield of the derivatization for nitrate and decreases the yield for nitrite due to hydrolysis. Yet, the use of the stable isotope analogs [ <sup>15</sup>N]nitrite and [15N]nitrate guarantees highly accurate quantitative measurements in biological samples [2]. −

**Scheme 1.** Derivatization of (**A**) nitrite and (**B**) nitrate in aqueous acetone with pentafluorobenzyl bromide at 50 ◦C. The optimum derivatization time is 5 min for nitrite and 60 min for nitrate.

Carbon dioxide (CO2) and carbonates are widely distributed in nature. In human blood, CO2/HCO<sup>3</sup> <sup>−</sup> is an important buffering system. In human urine, carbonate and bocrabonate are physiologically excreted [3,4]. Like nitrite and nitrate, we found that carbonate can react with PFB-Br under experimental conditions similar to those of nitrite and nitrate [5]. We observed the formation of the expected PFB-OCOOH derivative, albeit in low yield [5]. This behavior resembles in part that of nitrite derivatization with PFB-Br, which did not form any isolable PFB-ONO but exclusively (PFB-NO2).

In previous work, we observed an interaction of carbonate/bicarbonate with the analysis of nitrite in urine samples of subjects who took acetazolamide [6], a clinical drug [7]. Acetazolamide inhibits carboanhydrase (CA) activity in the kidneys, and because of this, it increases the excretion of bicarbonate and pH of the urine. Urine acidification of carbonatecontaining urine samples with acetic acid (20 wt.%) did not influence the derivatization of

nitrate but seemed to increase the yield of PFB-NO<sup>2</sup> [6]. In the present work, we investigated the derivatization of nitrite and nitrate in human urine and plasma under various conditions, aiming to reveal potential mechanisms and solutions for the derivatization of nitrite and nitrate in the presence of high carbonate/bicarbonate concentrations.

#### **2. Materials and Methods**

#### *2.1. Chemicals and Materials*

2,3,4,5,6-Pentafluorobenzyl bromide (PFB-Br), sodium nitrite (purity 99.99+%), sodium [ <sup>15</sup>N]nitrite and sodium [15N]nitrate (declared as 99 atom% at <sup>15</sup>N each) were obtained from Sigma-Aldrich (Steinheim, Germany). Toluene was purchased from Baker (Deventer, The Netherlands). Sodium bicarbonate and carbonate, acetone and glacial acetic acid were from Merck (Darmstadt, Germany). <sup>2</sup>H-Labelled creatinine ([*methylo*-<sup>2</sup>H3]creatinine, >99 atom% <sup>2</sup>H) was obtained from Aldrich. PFB-Br is corrosive and an eye irritant. Inhalation and contact with skin and eyes should be avoided. All work should be and was performed in a well-ventilated fume hood. Separate stock solutions of salts were prepared by dissolving accurately weighed amounts of commercially available unlabeled and stable-isotopelabeled salts in deionized water. Stock solutions were diluted with deionized water as appropriate.

Glassware for GC-MS (1.5-mL autosampler vials and 0.2-mL microvials) including the fused-silica capillary column Optima 17 (15 m × 0.25 mm I.D., 0.25-micrometer film thickness) were purchased from Macherey-Nagel (Düren, Germany).

#### *2.2. Derivatization Procedures for Nitrite and Nitrate and GC-MS Analyses*

In general, the following derivatization procedure was used. Deviations are reported in the individual experiments. A total of 100-µL aliquots of a sample were added to 400-µL aliquots of acetone and 10-µL aliquots of PFB-Br, and the samples were heated at 50 ◦C for 5 min or 60 min. After derivatization, acetone was removed under a stream of nitrogen, and analytes were extracted by vortexing with toluene (1 mL). Nitrite and nitrate were measured simultaneously in 100 µL urine specimens by a previously reported, fully validated GC-MS method immediately after acidification by using a 20 wt.% acetic acid solution and derivatization by PFB-Br, as described elsewhere [2]. <sup>15</sup>N-Labelled nitrite (final concentration, 4 µM or 1 µM) and <sup>15</sup>N-labeled nitrate (final concentration, 400 µM or 100 µM) were used as internal standards for urinary nitrite and nitrate, respectively. To investigate the effect of the CO2/Na2CO3/NaHCO<sup>3</sup> system on nitrite and nitrate analysis, samples were derivatized before and after acidification by 20 wt.% acetic acid to reach a final pH value of about 4.5 in order to remove CO<sup>2</sup> from urine samples, as described elsewhere [2]. Urinary excretion of nitrite and nitrate was corrected for urinary creatinine excretion. Creatinine-corrected excretion rates are reported as µmol of nitrite or nitrate per mmol of creatinine.

Aliquots (1 µL) of the toluene extracts were injected into the GC-MS apparatus (model DSQ from ThermoFisher; Dreieich, Germany) in the splitless mode. Quantification was performed by selected-ion monitoring (SIM) of mass-to-charge (*m*/*z*) of *m*/*z* 46 for [14N]nitrite, *m*/*z* 47 for [15N]nitrite, *m*/*z* 62 for [14N]nitrate and *m*/*z* 63 for [15N]nitrate using a dwell time of 50 ms for each ion. The measured peak area (PA) values of unlabeled and labeled nitrite and nitrate and the peak area ratio (PAR) of unlabeled to labeled nitrite or nitrate were used in calculations.

#### *2.3. Analyses in Urine Samples Collected in Previous Studies*

In the present study, we used urine samples collected in a previously reported study [6], which had been performed as follows in brief. Six apparently healthy volunteers (2 females, aged 25 and 44 years; 4 males, aged 24–49 years) had participated in the study. In the morning (8 a.m.), the volunteers were orally given one to two tablets acetazolamide (Acemit® 250 mg, medphano/Berlin, Germany) corresponding to a dose of about 5 mg/kg bodyweight. First, volunteers emptied their bladder and collected the

first urine specimen (time—2 h) followed by two collections at time—1 h and time 0 h. Immediately after collection of the 0 h urine sample, acetazolamide was taken by a glass of drinking water. Four urine samples were collected in polypropylene tubes in 30 min intervals and another four urine samples in 60 min intervals subsequently. Immediately after each collection, tubes were closed and put on ice. Urine samples were collected at several time points before and after acetazolamide ingestion, portioned in 1 mL and 10 mL aliquots and stored either at +5 ◦C for pH and carbonate measurement on the same day, or at −20 ◦C until analysis for nitrite, nitrate and creatinine on next day.

In some analyses, spot urine was collected by the author without any medication and used in some in the vitro studies on the effects of bicarbonate on nitrite and nitrate analyses.

#### *2.4. Statistical Analysis*

Results are expressed as mean with standard error of the mean, or as mean with standard deviation, as specified in the respective experiments. Differences between neighbor values were analyzed with paired or unpaired *t* tests as appropriate. *p* values ≤ 0.05 were considered statistically significantly different. Calculations and graphs were performed using GraphPad Prism version 7.0 (San Diego, CA, USA).

#### **3. Results**

#### *3.1. Effect of Exogenous Bicarbonate and Hydroxide on the Derivatization of [15N]nitrate, [ <sup>15</sup>N]nitrite and Endogenous Nitrate and Nitrite in Human Urine*

A healthy volunteer provided a morning urine. The freshly collected urine sample was spiked with 400 µM [15N]nitrate and 4 µM [15N]nitrite. This pooled urine sample was divided into two equal fractions that were spiked with freshly prepared 100 mM NaHCO<sup>3</sup> or 100 mM NaOH. Thereafter, each two 100 µL aliquots of the urine samples were derivatized with PFB-Br at 50 ◦C for varying times (0, 5, 10, 20, 30, 45 and 60 min) without prior acidification, as well as after acidification with 20 wt.% acetic acid. All nitrite and nitrate species were measured simultaneously by GC-MS using SIM of *m*/*z* 46 for [ <sup>14</sup>N]nitrite, *m*/*z* 47 for [15N]nitrite, *m*/*z* 62 for [14N]nitrate and *m*/*z* 63 for [15N]nitrate. The main results of this experiment are shown in Figure 1.

**Figure 1.** Effects of exogenous NaHCO<sup>3</sup> and NaOH on nitrite and nitrate analysis by GC-MS after derivatization with PFB-Br for the indicated times at 50 ◦C in a pooled human urine sample treated with 100 mM NaHCO<sup>3</sup> or with 100 mM NaOH. The samples were derivatized before and after acidification with 20 wt.% acetic acid. The concentrations of the internal standards in the urine samples were 400 µM [15N]nitrate and 4 µM [15N]nitrite. Data are shown from two independent experiments. All nitrite and nitrate species were measured simultaneously by GC-MS using selected-ion monitoring of (**A**) *m*/*z* 46 for [14N]nitrite and *m*/*z* 47 for [15N]nitrite, and of (**B**) *m*/*z* 62 for [14N]nitrate and *m*/*z* 63 for [15N]nitrate.

The PAR of *m*/*z* 46 to *m*/*z* 47 for nitrite and the PAR of *m*/*z* 62 to *m*/*z* 63 for nitrate behaved differently in the two urine samples. On the other hand, the PAR values behaved very similarly in the acidified urine samples that originally contained 100 mM NaHCO<sup>3</sup> or 100 mM NaOH. The greatest differences between the NaHCO3- and NaOH-treated urine samples were observed for nitrite. The PAR of *m*/*z* 46 to *m*/*z* 47 increased after 20 min of derivatization only in the non-acidified, NaHCO3-treated urine sample. This finding strongly indicates that it is not the alkalinity, but the CO2/Na2CO3/NaHCO<sup>3</sup> system of the urine that interferes with the analysis of nitrite in human urine. This interference can be eliminated by acidification of the urine sample, most likely by instantaneous conversion of Na2CO<sup>3</sup> and NaHCO<sup>3</sup> to highly volatile CO<sup>2</sup> [8].

#### *3.2. Effect of Exogenous Bicarbonate on the Derivatization of [15N]nitrate, [15N]nitrite and Endogenous Nitrate and Nitrite in Human Urine*

A pooled human urine sample was spiked with [15N]nitrate and [15N]nitrite at final concentrations of 100 µM and 1 µM, respectively. Each two 100 µL aliquots of this sample were spiked with an aqueous solution of NaHCO<sup>3</sup> to reach final added concentrations of 0, 20, 40, 60, 80 and 100 mM. After immediate derivatization with PFB-Br for 60 min, the derivatives were extracted with toluene (1 mL) and 1 µL aliquots thereof were analyzed by GC-MS in the SIM mode. Figure 2A shows that the peak area of [15N]nitrate increases linearly with increasing concentration of added NaHCO3, whereas the peak area of [15N]nitrite decreases at added NaHCO<sup>3</sup> concentrations of 60, 80 and 100 mM. Figure 2B shows that the peak area of endogenous nitrate (i.e., [14N]nitrate) increases linearly with increasing concentration of added NaHCO3, whereas the peak area of endogenous nitrite (i.e., [14N]nitrite) increases at added NaHCO<sup>3</sup> concentrations up to 60 mM with a tendency to slightly decrease at 80 mM and 100 mM NaHCO3. Figure 2C shows the PAR of *m*/*z* 46 to *m*/*z* 47 for nitrite and the PAR of *m*/*z* 62 to *m*/*z* 63 for nitrate. The PAR of *m*/*z* 62 to *m*/*z* 63 is constant, i.e., independent of the added NaHCO<sup>3</sup> concentration. The mean PAR *m*/*z* 62 to *m*/*z* 63 was 7.579 (RSD, 0.8%) corresponding to a concentration of 758 µM for endogenous nitrate in the urine sample. Previously, we found that the derivatization of nitrate with PFB-Br is incomplete even for 60 min at 50 ◦C [2]. The results of Figure 2 suggest that the formation of the PFB-O15NO<sup>2</sup> and PFB-O14NO<sup>2</sup> increases to the same extent in dependency on the NaHCO<sup>3</sup> concentration in the urine. Thus, nitrate can be reliably measured in human urine in the presence of high bicarbonate concentrations, as they may occur upon acetazolamide administration [6]. On the other hand, the results of Figure 2 indicate that NaHCO3, at concentration of 60, 80 and 100 mM, decreases the formation of PFB-15NO<sup>2</sup> and PFB-14NO2. This may eventually lead to overestimation and thus to inaccurate measurement of nitrite in urine samples that contain high concentrations of bicarbonate.

#### *3.3. Effects of Exogenous and Endogenous Bicarbonate, Acidification and Derivatization Time on the Derivatization of Nitrate and Nitrite in Human Urine*

Two pooled human urine samples, i.e., Urine X and Urine Y, were spiked with [ <sup>15</sup>N]nitrate and [15N]nitrite at the final added concentrations of 400 µM and 4 µM, respectively. Urine X was obtained from a volunteer who orally received acetazolamide. Urine Y was collected by another volunteer who did not receive any drug. The concentrations of bicarbonate, nitrate and nitrite were unknown in both urine samples. Urine Y was freshly spiked with 100 mM NaHCO3. Aliquots (100 µL) of the urines were derivatized with PFB-Br at 50 ◦C for different incubation times (range, 0 to 60 min) without and with acidification of the samples and analyzed by GC-MS as described above. The GC-MS chromatograms from these analyses are shown in Figure 3. The results of this experiment are illustrated in Figure 4.

**Figure 2.** Plots of the peak area of (**A**) *m*/*z* 63 for [15N]nitrate and *m*/*z* 47 for [15N]nitrite, of (**B**) *m*/*z* 62 for [14N]nitrate and *m*/*z* 46 for [14N]nitrite in a human urine sample spiked with the indicated bicarbonate concentrations, and of (**C**) the peak area ratio (PAR) of *m*/*z* 62 to *m*/*z* 63 for nitrate and of *m*/*z* 46 to *m*/*z* 47 for nitrite. The added concentrations were 100 µM for [15N]nitrate and 1 µM for [15N]nitrite. Data are shown from two independent experiments. The derivatization time with PFB-Br was each 60 min.

**Figure 3.** Representative partial GC-MS chromatograms from the simultaneous analysis of nitrite and nitrate in human urine samples without external addition of NaHCO<sup>3</sup> (**A**) and with addition of 100 mM NaHCO<sup>3</sup> (**B**). Derivatization with PFB-Br for 60 min at 50 ◦C in aqueous acetone was performed. The concentrations of the internal standards in the urine samples were 400 µM [15N]nitrate and 4 µM [15N]nitrite. GC-MS analysis was performed using selected-ion monitoring of *m*/*z* 46 for [14N]nitrite, *m*/*z* 47 for [15N]nitrite, *m*/*z* 62 for [14N]nitrate and *m*/*z* 63 for [15N]nitrate. The retention time (RT) was 2.95 min for nitrate and 3.12 min for nitrite. Note the decrease of the intensity and the peak area values of *m*/*z* 46 for [ <sup>14</sup>N]nitrite and *m*/*z* 47 for [15N]nitrite in presence of bicarbonate (**B**). AA means peak area calculated in the automated mode. The ThermoFisher quadrupole GC-MS apparatus model DSQ in the negative-ion chemical ionization mode was used.

**Figure 4.** Effects of exogenous bicarbonate and of the ingestion of acetazolamide on nitrate and nitrite analysis by GC-MS after derivatization with PFB-Br for the indicated times at 50 ◦C in two different human urine samples. The samples were derivatized before and after acidification with 20 wt.% acetic acid. The concentrations of the internal standards in the urine samples were 400 µM [15N]nitrate and 4 µM [15N]nitrite. Urine X (**A**,**C**,**E**,**G**) was collected upon ingestion of a 500-milligram tablet acetazolamide from a previous study [6]. Urine Y (**B**,**D**,**F**,**H**) was freshly collected by a healthy volunteer (author of the present article) and spiked with 100 mM NaHCO<sup>3</sup> . Data are shown from two independent experiments. All nitrite and nitrate species were measured simultaneously by GC-MS using selected-ion monitoring of *m*/*z* 46 for [14N]nitrite, *m*/*z* 47 for [15N]nitrite, (**B**) *m*/*z* 62 for [14N]nitrate and *m*/*z* 63 for [15N]nitrate. Left panels, Urine X; right panels, Urine Y. Acidified, close symbols; non-acidified, open symbols.

The effects of the derivatization time and acidification of the urine samples were qualitatively closely comparable. The peak area values of *m*/*z* 62 and *m*/*z* 63 increased with derivatization time and were lower in the acidified samples of Urine X and Urine Y (Figure 4A,B). The peak area values of *m*/*z* 46 and *m*/*z* 47 changed with derivatization time and were higher in the acidified samples of Urine X and Urine Y (Figure 4C,D). The highest peak area values were obtained at the derivatization time of 5 min. With the exception of the derivatization time of 0 min (urine sample was treated with PFB-Br and extracted immediately), the concentration of endogenous nitrate was independent of the derivatization time, but it was constantly lower in the acidified urine samples (Figure 4E,F). The concentration of endogenous nitrite was independent of the derivatization time of the acidified samples, but it increased constantly with the derivatization time larger than 10 min (Figure 4G,H).

#### *3.4. Effect of Exogenous Bicarbonate on the Derivatization of [15N]nitrite and Endogenous Nitrite in Human Plasma*

A pooled human plasma sample was spiked with [15N]nitrite at a final concentration of 1 µM. Each two 100-µL aliquots of this sample were spiked with an aqueous solution of NaHCO<sup>3</sup> to reach final added concentrations of 0, 10, 20, 40, 60, 80 and 100 mM. After derivatization with PFB-Br for 5 min, the derivatives were extracted with toluene and analyzed by GC-MS in the SIM mode. The results of these measurements are shown in Figure 5.

**Figure 5.** Plots of the peak area of (**A**) *m*/*z* 47 for exogenous nitrite (i.e., [15N]nitrite, 1 µM), of (**B**) *m*/*z* 46 for endogenous nitrite (i.e., [14N]nitrite) in a human plasma sample spiked with the indicated bicarbonate concentrations, and of (**C**) the resulting peak area ratio (PAR) of *m*/*z* 46 to *m*/*z* 47 for nitrite. Data are shown from two independent experiments. The derivatization time with PFB-Br was 5 min each. No [15N]nitrate was added to the plasma sample.

The peak area of *m*/*z* 47 for the internal standard [15N]nitrite and of *m*/*z* 46 for the endogenous nitrite ([14N]nitrite) decreased considerably at added NaHCO<sup>3</sup> concentrations of 10 mM, 20 mM and 40 mM (Figure 5A,B). However, the PAR of *m*/*z* 46 to *m*/*z* 47 was largely independent of the NaHCO<sup>3</sup> concentration (Figure 5C). The mean PAR was 1.357 (6.43%), indicating a concentration of 1.36 µM for endogenous nitrite in the plasma sample. These results suggest that the exogenous NaHCO<sup>3</sup> concentration equally inhibits the formation of PFB-15NO<sup>2</sup> and PFB-14NO<sup>2</sup> by maximally 50% under these conditions. Thus, the endogenous CO2/Na2CO3/NaHCO<sup>3</sup> system, which amounts to about 20 mM in total of human blood, is likely to inhibit the derivatization of nitrite with PFB-Br, yet without affecting analytical reliability in terms of accuracy. Unlike urinary nitrite, sample acidification is not pressingly needed in the quantitative analysis of nitrite in human plasma using PFB-Br derivatization.

#### **4. Discussion**

The derivatization of organic and inorganic anions such as nitrite, nitrate, chloride and carbonate is an indispensable analytical procedure in gas chromatography-based techniques for the vast majority of natural compounds. Pentafluorobenzyl bromide (PFB-Br) is a highly versatile derivatization reagent because of its favorable physicochemical properties with respect to both chromatography and detection due to its strongly electroncapturing F atoms [1]. The latter property leads to unbeatable amol-sensitivity in GC-MS-based approaches operating in the chemical ionization mode for numerous analytes in virtually all kinds of biological samples. As endogenous metabolites of NO and of environmental NO<sup>x</sup> species produced by human and natural activities, nitrite and nitrate are of general interest. Many different methods have been reported for the analysis of nitrite and nitrate in the last two centuries [9]. We found that endogenous nitrite and nitrate can be simultaneously derivatized with PFB-Br in numerous biological fluids and tissues in their acetonic solutions and suspensions [2]. Nitrite and nitrate are converted by PFB-Br to PFB-NO<sup>2</sup> and PFB-ONO2, respectively, virtually without the need of any catalyst. Nitrite and nitrate can be simultaneously quantitated by GC-MS as PFB-NO<sup>2</sup> and PFB-ONO<sup>2</sup> by using commercially available salts of [15N]nitrite and [15N]nitrate as internal standards, without problems arising from the need to chemically or enzymatically reduce nitrate to nitrite prior to derivatization.

PFB-Br is also suitable for the analysis of carboxylic groups-containing compounds in water-free organic solvents. Yet, the derivatization of fatty acids and their metabolites with PFB-Br requires the use of an organic base as a catalyst. Captopril is a carboxylic drug, and its derivatization with PFB-Br to its PFB ester has been reported to be catalyzed by carbonate [10]. Carbonate is often used in analytical derivatization, for instance, that of dimethylamine with pentafluorobenzoyl chloride, notably by means of extractive pentafluorobenzoylation [11,12]. However, the underlying mechanism of the catalytic action of carbonate is not yet fully understood. Previously, we had no indication that the derivatization of nitrite and nitrate with PFB-Br required carbonate/bicarbonate as a catalyst. We found that PFB-Br is suitable for the derivatization of carbonate/bicarbonate under the derivatization conditions of nitrite and nitrate [5]. In aqueous acetone, carbonate was found to form many reaction products. Two major carbonate derivatives were identified as CH3COCH2−C(OH)(OPFB)<sup>2</sup> and CH3COCH=C(OPFB)2, suggesting unique acetone-involving reactions. Two minor carbonate derivatives were PFB-OCOOH and O=CO2−(PFB)2. The GC-MS spectra of the PFB-O12COOH and PFB-O13COOH derivatives are shown in Figure 6 [5]. To the best of our knowledge, benzyl and pentafluorobenzyl esters of carbonate/bicarbonate have not been reported elsewhere, nor are they commercially available. PFB-OCOOH is presumably labile in aqueous solutions, but isolable by solvent extraction with toluene and apparently stable therein for GC-MS analysis [5]. It is proposed that PFB-OCOOH and O=CO2−(PFB)<sup>2</sup> are formed by the reaction of carbonate with one and two PFB-Br molecules, respectively, yet without the incorporation of acetone in these derivatives (Scheme 2).

**Figure 6.** Negative-ion chemical ionization GC-MS spectra of PFB-O12COOH and PFB-O13COOH, two minor derivatization products from the separate reaction of <sup>12</sup>C- and <sup>13</sup>C-carbonate in aqueous acetone with pentafluorobenzyl bromide (PFB-Br) for 60 min at 50 ◦C. PFB-O12COOH (M, 242.1) and PFB-O13COOH (M, 243.1) have the same retention time of 6.97 min. Inserts indicate the structure of the proposed anions of <sup>12</sup>C-carbonate (left, blue) and <sup>13</sup>C-carbonate (right, red). The anions *m*/*z* 167 and *m*/*z* 196 are derived from the derivatization reagent PFB-Br. These mass spectra have been previously reported as supplementary information in Ref. [5].

**Scheme 2.** Minor derivatization products from the reaction of carbonate in aqueous acetone with (**A**) one and (**B**) two pentafluorobenzyl bromide molecules at 50 ◦C. PFB-OCOOH (M, 241.1); PFB-O(CO)-O-PFB (M. 422.2).

In experiments with acetazolamide, a diuretic drug that massively enhances the excretion of carbonate/bicarbonate in the urine, we observed that the derivatization of nitrite with PFB-Br in urine samples of humans who ingested acetazolamide was decreased, suggesting a strong inhibitory effect of the conversion of nitrite into PFB-NO<sup>2</sup> [6]. This effect was abolished by acidifying the urine samples with aqueous acetic acid to pH values around 4.5 [6]. It is known that the derivatization of nitrite and halides with PFB-Br in its solutions in acetone, acetonitrile or ethanol are dependent on the pH value of the derivatization mixture [13]. It is also known that nitrite can react with CO<sup>2</sup> [14]. Thus, the CO2/Na2CO3/NaHCO<sup>3</sup> system can interfere with the PFB-Br derivatization of nitrite and nitrate in biological samples by several different mechanisms. In the present work, we addressed such potential mechanisms.

In order to gain more mechanistic information in this study, we used [15N]nitrite and [15N]nitrate, human urine and plasma samples spiked with NaHCO<sup>3</sup> in relevant physiological and pharmacological concentration ranges. We also used urine samples from volunteers who took acetazolamide at pharmacological doses (around 5 mg/kg bodyweight).

The results of the present and previous studies suggest that carbonate does not catalyze the derivatization of nitrate to PFB-ONO<sup>2</sup> with PFB-Br in homogenous phase in aqueous acetone. Carbonate/bicarbonate-containing biological samples, notably human urine, are by nature alkaline. However, it is not the alkalinity itself but the presence of carbonate/bicarbonate at high concentrations that is responsible in part for the massive impairment of nitrite derivatization with PFB-Br. The concentration of carbonate/bicarbonate in human plasma is of the order of 20 mM, that of nitrate in the range of 20 to 100 µM and that of nitrite of the order of 1 µM. The concentration of carbonate/bicarbonate in human urine is usually below 10 mM, that of nitrate of the order of 1000 µM and that of nitrite of the order of 10 µM. In the case of acetazolamide ingestion, blood carbonate/bicarbonate and blood pH only slightly change. However, in urine, carbonate/bicarbonate can reach concentrations up to about 100 mM upon acetazolamide intake, whereas nitrate and nitrite concentrations in urine instead decrease due to the diuretic effects of the drug. Thus, in such urine samples, carbonate/bicarbonate are present in a high molar excess of nitrate and especially of nitrite. As an example, we considered a urine sample that contains 10 mM carbonate/bicarbonate, 1000 µM nitrate and 10 µM nitrite. Under regular derivatization conditions (100 µL urine, 10 µL PFB-Br equivalent to 70 µmol), PFB-Br is present in a very high molar excess in carbonate/bicarbonate (69:1 µmol), nitrate (690:1) and nitrite (6900:1). In addition, human urine contains many other organic and inorganic substances at mM concentrations, such as creatinine [15] and chloride [16], which can react with PFB-Br under the same derivatization conditions. Thus, nitrate and nitrite compete with many other species for PFB-Br. High increases of the concentration of competing nucleophiles with small changes of nitrate and nitrite concentrations, for instance carbonate/bicarbonate, would decrease the molar ratio of PFB-Br to nitrate and nitrite. As the yield of derivatization reactions with PFB-Br also depends upon the PFB-Br concentration [13], high increases of competitive analytes would decrease the derivatization yield of PFB-NO<sup>2</sup> and PFB-ONO2. Our studies show that increasing carbonate/bicarbonate concentrations decrease the yield of PFB-NO2, but they increase the yield of PFB-ONO2. One may therefore conclude that competition alone cannot explain the opposite effects of carbonate/bicarbonate on nitrate and nitrite derivatization with PFB-Br.

A more convincing assumption could be that intermediate derivatives of carbonate/bicarbonate with PFB-Br, notably PFB-OCOOH and PFB-O(CO)-O-PFB, interact with nitrate and nitrite to produce diametrically opposed effects on the conversion of nitrate to PFB-ONO<sup>2</sup> and of nitrite to PFB-NO<sup>2</sup> (Scheme 3). The nucleophilic attack of nitrate on the benzyl groups of PFB-OCOOH and PFB-O(CO)-O-PFB would then increase the formation of PFB-ONO2, thereby releasing carbonate. As PFB-ONO has not been detected thus far, it is possible that nitrite attacks the benzyl group of PFB-Br exclusively with its N atom to produce to PFB-NO2. This reaction occurs more rapidly and abundantly than the reaction

of nitrate with PFB-Br to generate PFB-ONO<sup>2</sup> [2]. That carbonate/bicarbonate inhibited the formation of PFB-NO<sup>2</sup> suggests that nitrite cannot react with PFB-OCOOH and PFB-O(CO)- O-PFB to form PFB-NO<sup>2</sup> and to release carbonate. Yet, it is also possible that carbonate reacts with PFB-NO<sup>2</sup> and PFB-ONO<sup>2</sup> to generate PFB-OCOOH, thereby releasing nitrite and nitrate, respectively (Scheme 3). Thus, the reaction of carbonate/bicarbonate with PFB-NO<sup>2</sup> is considered irreversible and decreases the concentration of PFB-NO<sup>2</sup> during the derivatization. On the other hand, the reaction of carbonate/bicarbonate with PFB-ONO<sup>2</sup> is considered reversible and eventually increases the concentration of PFB-ONO<sup>2</sup> during the derivatization, in addition to the direct reaction of nitrate with PFB-Br.

**Scheme 3.** Proposed reactions for the nucleophilic substitution by carbonate from (**A**) PFB-NO<sup>2</sup> and (**B**) PFB-ONO<sup>2</sup> to release nitrite and nitrate, respectively, during the derivatization in aqueous acetone with pentafluorobenzyl bromide at 50 ◦C in the presence of carbonate. Reaction (**A**) is considered irreversible, reaction B is assumed reversible.

In theory, carbonate/bicarbonate may contribute to nitrate in the case of chromatographic co-elution of PFB-OCOOH and PFB-ONO2. This is because the <sup>13</sup>C isotope of carbonate forms PFB-O13COOH, which ionizes to form *m*/*z* 62, albeit to a very low extent of about 2% (Figure 6). Such a contribution is considered very low because of the natural abundance of <sup>13</sup>C of 1.1%. Nevertheless, the contribution of carbonate/bicarbonate to nitrate may be higher in the case that <sup>13</sup>C-carbonate is used as the internal standard for endogenous carbonate/bicarbonate.

#### **5. Conclusions**

The derivatization of inorganic anions such as nitrite, nitrate and carbonate/bicarbonate in biological and environmental samples with PFB-Br using acetone as the organic solvent is performed in the homogenous phase and makes their GC-MS analysis possible. Although quantitative analysis is realized by using stable isotope labeled analogs, such as <sup>15</sup>N-nitrite, <sup>15</sup>N-nitrate and <sup>13</sup>C-carbonate, interferences may occur due to their ubiquitous occurrence in the form of their contaminants. Another potential source of interference with the analysis of nitrite and nitrate in human urine may be the occurrence of carbonate/bicarbonate at manifold higher concentrations than under normal conditions. We identified such a condition when humans ingested pharmacological doses of the diuretic acetazolamide, an inhibitor of human CA II and CA IV. Acetazolamide potently inhibits renal CA activity and, in this way, massively increases the excretion of carbonate/bicarbonate in the urine and alkalizes the urine to pH values of about 8. Our studies indicate that carbonate/bicarbonate

exerts diametrically opposed effects on the analysis of nitrite and nitrate by GC-MS when derivatized with PFB-Br. Carbonate/bicarbonate increases the formation of PFB-ONO<sup>2</sup> but decreases the formation of PFB-NO2. These effects are not due to the concurrent alkalization of the urine by drugs such as acetazolamide. Rather, carbonate/bicarbonate increases the formation of PFB-ONO<sup>2</sup> by enhancing the reaction of nitrate with an intermediate and isolable reaction product of carbonate/bicarbonate with PFB-Br, i.e., PFB-OCOOH. Unlike in other derivatization reactions such as with pentafluorobenzoyl chloride, carbonate/bicarbonate does not act as a catalyst in the derivatization of nitrate and nitrite with PFB-Br. On the other hand, carbonate/bicarbonate decreases the formation of PFB-NO<sup>2</sup> most likely due the inability of nitrite to attack the PFB-OCOOH via its N atom. Such effects are much less pronounced in human plasma, in part because of the lower carbonate/bicarbonate concentration and in part due to the higher buffer capacity of the plasma compared to urine. A very simple and effective solution of the negative effect of high carbonate/bicarbonate concentrations on nitrite measurement in urine as PFB-NO<sup>2</sup> is mild acidification by adding small volumes of 20 wt.% acetic acid to the urine.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Ethical review and approval were waived for the present work, due to the use of human urine samples originally collected in a previous study cited in this work (i.e., Ref. [6]).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The technical assistance of Anja Mitschke is gratefully acknowledged.

**Conflicts of Interest:** The author declares no conflict of interest.

**Sample Availability:** Samples of the compounds are not available from the authors.

#### **References**


### *Article* **Structural Characterization of Unusual Fatty Acid Methyl Esters with Double and Triple Bonds Using HPLC/APCI-MS <sup>2</sup> with Acetonitrile In-Source Derivatization**

**Petra Horká 1,2, Vladimír Vrkoslav 1 , Jiˇrí Kindl 1 , Karolina Schwarzová-Pecková <sup>2</sup> and Josef Cvaˇcka 1,2, \***


**Abstract:** Double and triple bonds have significant effects on the biological activities of lipids. Determining multiple bond positions in their molecules by mass spectrometry usually requires chemical derivatization. This work presents an HPLC/MS method for pinpointing the double and triple bonds in fatty acids. Fatty acid methyl esters were separated by reversed-phase HPLC with an acetonitrile mobile phase. In the APCI source, acetonitrile formed reactive species, which added to double and triple bonds to form [M + C3H5N] +• ions. Their collisional activation in an ion trap provided fragments helpful in localizing the multiple bond positions. This approach was applied to fatty acids with isolated, cumulated, and conjugated double bonds and triple bonds. The fatty acids were isolated from the fat body of early-nesting bumblebee *Bombus pratorum* and seeds or seed oils of *Punicum granatum*, *Marrubium vulgare*, and *Santalum album*. Using the method, the presence of the known fatty acids was confirmed, and new ones were discovered.

**Keywords:** acetonitrile-related adducts; acetylenic lipids; double and triple bond localization; in-source derivatization; mass spectrometry

#### **1. Introduction**

The localization of double and triple bonds (DBs and TBs) is a key step in the structural characterization of fatty acids (FAs). The biological functions of lipids are often linked to the specific arrangement of multiple bonds in their FA chains. Lipids with unusually arranged double bonds and triple bonds are of interest because of their function in living organisms and their unique biological effects and potential use in medicine [1,2]. Mass spectrometry is useful for pinpointing the double bonds in FAs and their methyl esters (FAMEs), especially when combined with chromatography. The early methods were based on the electron ionization of derivatized lipids. Derivatization, either at the double bond site or at the carboxylic group, is required because of the bond migration along the aliphatic chains during electron ionization. Numerous FA derivatives, including pyrrolidides [3], 4,4-dimethyloxazoline (DMOX) [4], or dimethyl disulfide (DMDS) derivatives [5], have found their use in GC/MS. Later, HPLC/MS-based methods began to be developed. Unlike GC/MS, these methods also make it possible to analyze less volatile and non-volatile FAs and their derivatives. A number of methods have been proposed for localizing double bonds using electrospray ionization, including Paternò–Büchi photochemical derivatizations [6–8], epoxidation in low-temperature plasma [9,10] and negative-ion paper-spray ionization [11], post-column epoxidation and peroxidation [12], charge-switch derivatization with *N*-(4-aminomethylphenyl) pyridinium (AMPP) [13,14], or ozone-induced dissociation (OzID) [15,16], and combining charge-switch derivatization with OzID [17].

**Citation:** Horká, P.; Vrkoslav, V.; Kindl, J.; Schwarzová-Pecková, K.; Cvaˇcka, J. Structural Characterization of Unusual Fatty Acid Methyl Esters with Double and Triple Bonds Using HPLC/APCI-MS<sup>2</sup> with Acetonitrile In-Source Derivatization. *Molecules* **2021**, *26*, 6468. https://doi.org/ 10.3390/molecules26216468

Academic Editor: Paraskevas D. Tzanavaras

Received: 21 September 2021 Accepted: 21 October 2021 Published: 26 October 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Besides electrospray ionization, atmospheric-pressure chemical ionization (APCI) can also be applied for localizing double bonds in HPLC/MS [18–23]. The methods rely on acetonitrile-related reactive species formed in the ion sources. The use of even-electron (1-methyleneimino)-1-ethenylium as a reagent for derivatizing double bonds was initially developed for chemical ionization [24–27] and later applied in APCI-MS [18]. Using helium as a nebulizing gas, C3H4N<sup>+</sup> adducts ([M + 54]<sup>+</sup> ) of triacylglycerols (TGs) were formed, and their CID spectra indicated the positions of the original double bonds [18]. Later, we showed that APCI sources operated under conventional conditions with nitrogen nebulizing gas yield odd-electron C3H5N+• adducts ([M + 55]+• ) [19]. The collision activation of the adducts induced cleavages of C–C bonds next to the original double bond, leading to pairs of diagnostic fragments indicating the double bond position. The advantage of this approach lies in its simplicity: the only requirement for an HPLC/APCI-MS<sup>2</sup> method is the presence of acetonitrile in the mobile phase. The method has been applied for the structure elucidation of various unsaturated lipids, including FAMEs [20,28], hydroxy-FAMEs [23], wax esters [19], diol diesters [22], or TGs [21].

To date, only a few methods for determining the position of triple bonds in lipids have been published [27,29–32]. Triple bonds in FAs can be pinpointed after DMOX derivatization using GC/MS [31]. While a conjugated system of double bonds manifests itself by a series of fragments differing by 12 Da, triple bond-related fragments differ by 10 Da. It allows for the structural characterization of conjugated ene–yne acids. Still, the fragmentation of conjugated yne–yne or yne–yne–ene bonds is more complex, and the spectra are difficult to interpret [30]. Using this approach, many acetylenic lipids have been identified in plants [29,30,32]. The position of a triple bond can also be determined using acetonitrile chemical ionization based on (1-methyleneimino)-1-ethenylium adducts formation [27]. To the best of our knowledge, no method for localizing triple bonds using HPLC/MS has appeared in the literature so far.

Double bond positions in FAs reflect specificities of desaturases involved in their biosynthesis. Most monounsaturated FAs have a double bond in 9-position. Other positions are also relatively common, for instance, 7-position in algae, 5- and 10-positions in bacteria, or 6-position in plants [33]. Double bonds in polyunsaturated FAs are typically spaced by one methylene group (methylene interrupted). FAs with double bonds separated by two or more methylene units are found, for instance, in marine sponges *Microciona prolifera* (FA 26:2n-17,21 and FA 26:3n-7,17,21) [34,35], *Dysidea fragilis* (FA 25:3n-8,16,20; FA 25:3n-6,16,20; FA 24:3n-7,15,19 and FA 24:2n-7,17) [36], or *Hymeniacidon sanguinea* (e.g., FA 28:2n-9,19,23; FA 26:2n-17,21; FA 26:3n-7,17,21; FA 24:2n-15,19 and FA 24:3n-7,15,19) [37]. More than twenty different FAs with double bonds separated by two or more methylene units were identified in the gonads of limpets *Cellana grata* [38], *Collisella dorsuosa* [38], and *Cellana toreum* [39,40]. Unusual FAs with 24, 26, and 28 carbon atoms were found in TGs isolated from the fat body of early-nesting bumblebee *Bombus pratorum*. FA 26:2n-7*c*,17*c* occupied one, two, or all three positions in the TGs [41].

FAs with a conjugated system of double bonds are mostly represented by conjugated linoleic acids (CLAs) and conjugated linolenic acids (CLnAs), which are collective terms for the positional and geometric isomers of octadecadienoic and octadecatrienoic acids, respectively. CLAs exist naturally at higher concentrations in animal products, such as milk fat, cheese, and ruminant meat [42,43]. Two double bonds in CLAs are primarily in positions 9 and 11, or 10 and 12, and each of the double bonds can be either *cis* or *trans* [44]. CLAs are important for human nutrition. For instance, *cis*-9,*trans*-11 and *trans*-10,*cis*-12 isomers reduce carcinogenesis and atherosclerosis, increase bone and muscle mass, and exhibit antidiabetic effects [42,45]. CLnAs are found in plant seed oils, including oils from *Vernicia fordii* (α and β-eleostearic acid; FA 18:3n-5*t*,7*t*,9*c* and FA 18:3n-5*t*,7*t*,9*t*) [46], *Catalpa bignonoides* (catalpic acid; FA 18:3n-5*c*,7*t*,9*t*) [47], *Jacaranda mimosifolia* (jacaric acid; FA 18:3n-6*c*,8*t*,10*c*) [48], or *Calendula officinalis* (alfacalendic acid; FA 18:3n-6*c*,8*t*,10*t* and beta-calendic acid; FA 18:3n-6*t*,8*t*,10*t* [49]. A rich source of CLnA is pomegranate (*Punicum granatum*) seed oil (PSO). It contains punicic

acid (FA 18:3n-5*c*,7*t*,9*c*), α-eleostearic acid (18:3n-5*t*,7*t*,9*c*), β-eleostearic acid (18:3n-5*t*,7*t*,9*t*), and catalpic acid (18:3n-5*c*,7*t*,9*t*) [50–54]. The structures of several other CLnAs in PSO remain to be clarified [51,55]. CLnAs are known for their antioxidant, anti-inflammatory, anti-atherosclerotic, antitumor, and serum lipid-lowering activities. They help fight against cancers, obesity, diabetes, and heart diseases [53,56,57].

Lipids with two cumulated double bonds (allenic lipids) are found in Lamiaceae family plants; elsewhere in nature, they are rare [58,59]. The first known C18 allenic FA, laballenic acid (FA 18:2n-12,13), was isolated from *Leonotis nepetaefolia* seed oil [60] and later reported also from other Lamiaceae species [61]. Lamenallenic acid (FA 18:3n-2*t*,12,13) was discovered in *Lamium purpureum* seed oil [62]. Phlomic acid (FA 20:2n-12,13) was found in several species of *Phlomis* genus (Lamiaceae) [61]. Seeds of *Marrubium vulgare* contain laballenic acid and phlomic acid [59]. Allenic lipids are known for their anticancer, anti-inflammatory, antiviral, and antibacterial activities [1].

FAs with triple bonds (acetylenic FAs) are relatively widely distributed in nature. They are found in plants, fungi, microorganisms, and invertebrates [58,63–65]. FAs and other acetylenic lipids in plants serve as chemical protection against microorganisms. They are toxic to bacteria, viruses, and insects [2,66–69]. Many acetylenic lipids exhibit fungicidal, phototoxic, antitumor, and other properties [1], which render them potentially useful in medicine. The chain length and triple bond positions affect their fungicidal properties [2,68]. The structures and cytotoxic activities of acetylenic lipids were reviewed recently [70]. Some plant FAs contain triple and double bonds conjugated, e.g., pyrulic acid (FA 17:2n-7,9TB), ximenynic (also termed santalbic) acid (FA 18:2n-7*t*,9TB), or heisteric acid (FA 18:3n-7*t*,9TB,11*c*) from *Heisteria silvanii* seed oil [32]. *Ximenia americana* contains FA 18:1n-13TB and FA 18:4n-2,4,8,6TB [2,66]. Santalbic acid (FA 18:2n-7*t*,9TB), identified for the first time in *Santalum album* [58,63], is one of the few acetylenic FAs occurring at higher levels in plants. It is found in the seed oils of the Santalaceae, Olacaceae, and Opiliaceae families, where it can reach up to 95% of the total FAs [71,72]. Other biologically active acetylenic acids are crepenynic acid (FA 18:2n-6TB,9*c*), tariric acid (FA 18:1n-12TB), stearolic acid (FA 18:1n-9TB), or nonadec-6-ynoic acid (FA 19:1n-12 TB) [67,73–75]. FAs with a triple bond can also be found in water mosses [40,76–79].

This work deals with the localization of double and triple bonds in FAMEs. The conversion of lipids or lipid mixtures to FAMEs is frequently used in lipidomics workflows because the GC or LC analysis of FAMEs provides quick and valuable information on the fatty acyl chains. Here, FAME standards and FAMEs obtained by the transesterification of the TGs from biological samples were analyzed by HPLC/APCI-MS/MS using an acetonitrile mobile phase. Isolated, cumulated, and conjugated double bonds and triple bonds were localized using the fragmentation of [M + C3H5N]+• adducts generated in the ion source. To the best of our knowledge, the localization of triple bonds in FAMEs by RP-HPLC with MS detection is reported here for the first time.

#### **2. Results and Discussion**

The chromatographic separation of FAMEs was achieved on the Develosil RP-Aqueous C30 column using isocratic elution with acetonitrile. The mobile phase in the APCI source formed reactive species, which added to double and triple bonds. The adducts were isolated and activated in the ion trap to generate ions bearing information on the original double or triple bond position. The diagnostic ions formed by the cleavages of adjacent C–C bonds were marked α if they carried the ester moiety or ω if they contained the terminal-carbon end without the ester group. The diagnostic peaks corresponding to cleavages before the first and after the last unsaturated bond in polyunsaturated FAMEs tended to be more abundant than the others. This phenomenon was used for deducing the arrangement of the double and triple bonds in polyunsaturated chains. A parameter named "multiple bond region" (MBR) was calculated and tabulated for various theoretically possible arrangements of multiple bonds (Table 1). The MBR value was calculated using theoretical *m/z* values of the adduct (precursor) and α and ω fragments corresponding to cleavages before the first and after the last unsaturated bond as follows:

$$\text{MBR} = m/z \left( \mathfrak{a} \right) + m/z \left( \omega \right) - m/z \left( \left[ \mathbf{M} + 5\mathbf{5} \right]^{+\bullet} \right) \tag{1}$$

**Table 1.** Multiple bond region (MBR) values for common arrangements of double bonds (DBs) and triple bonds (TBs) in polyunsaturated chains.


The experimental MBR values calculated for the adduct and the most abundant α and ω fragments in the spectra were then compared to theoretical MBRs. For instance, the MS/MS spectrum of [M + 55]+• adduct of unknown FA at *m/z* 347.0 provided the most abundant α and ω peaks at *m/z* 290.2 and *m/z* 190.2, respectively. The calculated MBR value (290 + 190 − 347 = 133) suggested FAME with three conjugated double bonds (Table 1). Diagnostic ions were accompanied by less abundant satellite peaks differing from α and ω ions by 14 or 15 Da. These fragments representing cleavages at more distant C–C bonds were important for distinguishing double and triple bonds. The elemental composition of the major fragments in the spectra of FAME standards was confirmed by Orbitrap high-resolution data (Supplementary Materials Table S1).

#### *2.1. Mass Spectra of Standards with Conjugated Double Bonds*

The system with two conjugated double bonds was investigated using standards of FAME 18:2n-7*t*,9*t* (Mangold's acid methyl ester) and FAME 18:2n-7*c*,9*c* (ricinenic acid methyl ester). The fragments in the MS/MS spectrum for FAME 18:2n-7*t*,9*t* (Figure 1) were rationalized as follows: α n-7 peak at *m/z* 264.1, α n-9 peak at *m/z* 238.2, ω n-7 peak at *m/z* 166.1, and ω n-9 peak at *m/z* 192.1. The MBR value calculated from the two most intense fragments in the spectrum (i.e., *m/z* 192.1 and *m/z* 264.1) was 107. Despite the presence of satellite fragments differing by 14 Da from the diagnostics peaks, the spectrum provided clear evidence of two conjugated double bonds in the n-7 and n-9 positions. The spectrum of FAME 18:2n-7*c*,9*c* having the opposite geometry on both double bonds looked similar (Figure S1), which confirmed the negligible effect of double bond geometry on the adduct fragmentation documented earlier [19].

The MS/MS spectrum of punicic acid methyl ester with three conjugated double bonds (FAME 18:3n-5*c*,7*t*,9*c*) is shown in Figure 2. The major fragments in the spectrum were formed by cleavages before and after the series of double bonds. They were easily distinguishable from the other ions. The most abundant fragments α n-5 at *m/z* 290.2 and ω n-9 at *m/z* 190.2 delimited the group of conjugated double bonds and corresponded to an MBR value of 133. The fragments formed by the cleavages between conjugated double bonds α n-7 (*m/z* 264.3), α n-9 (*m/z* 238.2), ω n-7 (*m/z* 164.2), and ω n-5 (*m/z* 138.2) were of low intensities but discernable in the spectrum. The same diagnostic fragments and MBR value could theoretically be expected for a FAME with two cumulated double bonds separated by one methylene group from the third double bond. Such an arrangement of double bonds would be, however, clearly distinguishable because the system of cumulated double bonds manifests itself by abundant α + 1 Da ion (Section 2.3.3). Such an ion (*m/z* 251 or *m/z* 291 in this case) is not present in the spectrum. Therefore, the spectrum in Figure 2 can be unambiguously interpreted as FAME 18:3n-5,7,9. −

− **Figure 1.** APCI MS/MS CID spectrum of [M + 55]+• adduct of Mangold's acid methyl ester (FAME 18:2n-7*t*,9*t*); MBR = 264 + 192 − 349 = 107.

**Figure 2.** APCI MS/MS CID spectrum of [M + 55]+• adduct of punicic acid methyl ester (FAME 18:3n-5*c*,7*t*,9*c*); MBR = 290 + 190 − 347 = 133.

#### − *2.2. Mass Spectra of Standards with a Triple Bond*

−

ω

Figure 3 shows the MS/MS spectrum of FAME 18:1n-9TB (stearolic acid methyl ester) [M + 55]+• adduct. The abundant fragments *m/z* 236.2 (α n-9TB) and *m/z* 192.2 (ω n-9TB) clearly indicated a triple bond in the n-9 position. Unlike FAMEs with double bonds, the satellite fragments differed by +15 Da from α TB and ωTB (*m/z* 207.1 and *m/z* 251.1, respectively). The intensities of the diagnostic fragments and their +15 Da satellites were similar, allowing us to recognize these peaks in the spectrum easily. Such a pattern distinctly indicated a triple bond. Satellite fragments differing by +14 Da, typical for double bonds, were present at significantly lower intensities.

**Figure 3.** APCI MS/MS CID spectrum of [M + 55]+• adduct of stearolic acid methyl ester (FAME 18:1n-9 TB); MBR = 236 + 192 − 349 = 79.

α ω

α

α ω

− ω The satellite fragment ions made it also possible to characterize FAMEs with a combination of double and triple bonds. For instance, crepenynic acid methyl ester with one double bond and one triple bond (FAME 18:2n-6TB,9c) provided a spectrum with the most abundant peak at *m/z* 150.1 (Figure 4). This signal is a diagnostic fragment for triple bond (ω n-6TB) because its satellite appears at a 15 Da higher *m/z* value (*m/z* 165.0). Analogously, the *m/z* 276.1 with its satellite at *m/z* 291.1 is the triple bond diagnostic peak (α n-6TB). Fragment *m/z* 190.1 indicates a double bond (ω n-9) because its satellite peak appears at *m/z* 204.1. α ω

− **Figure 4.** APCI MS/MS CID spectrum of the [M + 55]+• adduct of crepenynic acid methyl ester (FAME 18:2n-6TB,9c); MBR = 276 + 190 − 347 = 119.

#### *2.3. Analysis of Natural Samples*

The fragmentation of FAME standards with various arrangements of double and triple bonds helped us characterize the FAMEs isolated from biological samples. The identification procedure was initiated by deducing the number of carbons and level of unsaturation from the *m/z* values of the protonated FAMEs. The second step examined the

MS/MS spectra of [M + 55]+• ions to identify the diagnostic fragments and their satellites. The *m/z* values of the diagnostic fragments, MBR values, and the mass difference between the diagnostic fragments and satellites were used to deduce the positions of double and triple bonds. Finally, the retention times were checked for their consistency with the expected elution order of FAMEs [80,81].

α ω

#### 2.3.1. FAMEs from the Fat Body of *Bombus pratorum*

The early-nesting bumblebee *Bombus pratorum* is widespread in Europe. It is one of the earliest bumblebee species to emerge from hibernation each year. The fat body of *B. pratorum* males contains TGs with long, diunsaturated fatty acyls, which are structurally related to its marking pheromone [41].

The chromatogram of *B. pratorum* FAMEs is shown in Figure 5. The MS/MS spectra of diunsaturated FAMEs (Figure 6) provided abundant and recognizable α and ω fragments interpreted as FAME 24:2n-7,17, FAME 25:2n-7,17, and 26:2n-7,17. The double bond positions were in excellent agreement with previous work, where the positions of the double bonds were established using dimethyl disulfide derivatization [41].

Altogether, nine saturated, fourteen monounsaturated, five diunsaturated, and one triunsaturated FAMEs were detected (Table 2). Nine of them (FAME 17:1n-7; FAME 17:0; FAME18:2n-3,6; FAME 18:1n-8; FAME 19:1n-7; FAME 19:0; FAME 22:1n-7; FAME 23:0; FAME 25:2n-7,17) are reported here for *B. pratorum* for the first time. To the best of our knowledge, FA 25:2n-7,17 has not been mentioned in the literature so far. FAMEs 25:2 are very rare in nature; the only known source of such acids are marine sponges producing different isomers [37,82–84]. FA 25:2n-7,17 likely serves as a precursor for tetracosadiene, a minor component (0.02 to 0.3%; I. Valterová 2021, personal communication, 18 April) of *B. pratorum* males' secretion. Bumblebee males use the secretion to mark their patrolling routes [85]. −

**Figure 5.** HPLC/APCI-MS base-peak chromatogram of FAMEs from the fat body of *Bombus pratorum* and the list of identified species.

**Figure 6.** APCI MS/MS spectra of the [M + 55]+• adducts of FAME from *B. pratorum* interpreted as FAME 24:2n-7,17 (**A**), 25:2 n-7,17 (**B**), and 26:2n-7,17 (**C**).


**Table 2.** FAMEs identified in TG fraction of *B. pratorum* fat body lipids.

\* Mean ± SD values of relative peak area values obtained by integrating GC/MS peaks; data for five bumblebee individuals. From ref. [41].

#### 2.3.2. FAMEs from Pomegranate Seed Oil

Pomegranate (*Punicic granatum*) seed oil (PSO) is a rich source of FAs with conjugated double bonds. Cold-pressed PSO was transesterified, and the resulting mixture was analyzed by HPLC/MS. Many isomeric species with similar retention times tended to coelute. Still, the partial separation of the peaks allowed us to identify most of these lipids (Figure 7).

All the abundant peaks corresponded to CLnAs. The highest intensity exhibited an isomer with t<sup>R</sup> 11.2 min, which was interpreted as FAME 18:3n-5,7,9. Its MS/MS spectrum (Figure 8A) showed abundant diagnostic peaks *m/z* 190.2 and *m/z* 290.1, corresponding to an MBR value of 133. The spectrum closely matched the punicic acid methyl ester shown in Figure 2. Interestingly, two less abundant isomers with the same diagnostic fragments were detected at t<sup>R</sup> 12.0 min and t<sup>R</sup> 14.8 min (Supplementary Materials Figure S2). These species were isomers with the same double bond positions but different double bond geometries. The geometrical isomers of punicic acid, namely FAME 18:3n-5*t*,7*t*,9*c* (β-eleostearic acid); FAME 18:3n-5*c*,7*t*,9*t* (α-eleostearic acid), and FAME 18:3n-5*t*,7*t*,9*t* (catalpic acid) were detected in pomegranate seed oil previously [51,86–89]. As the elution of the FAs in reversed-phase systems proceeds from *cis* to *trans* isomers [20,90], the later eluting isomers likely contained a higher number of *trans* double bonds. The MS/MS spectra of FAMEs with three conjugated double bonds in different positions are shown in Figure 8B–F. In all of them, the MBR value was 133, and the diagnostic fragments allowed us to interpret them as

FAME 18:3n-4,6,8 (Figure 8B), FAME 18:3n-3,5,7 (Figure 8C), FAME 18:3n-2,4,6 (Figure 8D), FAME 18:3n-8,10,12 (Figure 8E), and FAME 18:3n-9,11,13 (Figure 8F). The retention times of the latter two CLnAs were close to each other, which resulted in mixed spectra. Overall, ten CLnAs, one methylene-interrupted (18:2n-6,9), and two monounsaturated (18:1n-9 and 20:1n-9) FAMEs were identified in the PSO (Table 3). The results were in good agreement with previous analyses of PSO by silver-ion HPLC [51]. HPLC-based approaches to CLnAs analysis offer a higher number of isomers detected than GC [55,88,91,92]. We found four new CLnAs in the PSO, which, to the best of our knowledge, have not yet been described in the literature: two geometric isomers of 18:3n-2,4,6 (Figures 8D and S2), FAME 18:3n-8,10,12 (Figure 8E), and FAME 18:3n-9,11,13 (Figure 8F). They are characterized by the double bonds closer to the terminal carbon end (FAME 18:3n-2,4,6) or methyl ester group (FAME 18:3n-8,10,12 and FAME 18:3n-9,11,13).


**Table 3.** FAMEs identified in TG fraction of pomegranate seed oil.

**Figure 7.** HPLC/APCI-MS base-peak chromatogram of FAMEs obtained from *Punicum granatum* seed oil and the list of identified species.

α

**Figure 8.** APCI MS/MS spectra of the [M + 55]+• adducts of selected conjugated FAMEs from PSO interpreted as FAME 18:3n-5,7,9 (**A**), 18:3n-4,6,8 (**B**), 18:3n-3,5,7 (**C**), 18:3n-2,4,6 (**D**), 18:3n-8,10,12 (**E**), and 18:3n-9,11,13 (**F**).

#### 2.3.3. FAMEs from *Marrubium vulgare* Seeds

White horehound (*Marrubium vulgare*) is a perennial, aromatic herb native to Europe, northern Africa, and southwestern and central Asia. Like other plants of the Lamiaceae family, it contains FAs with cumulated double bonds (allenic FAs). TGs from white horehound seeds were transesterified, and the resulting mixture of FAMEs analyzed by HPLC/MS (Figure 9). FAMEs with 18 to 21 carbons and up to three double bonds were detected.

β

The most abundant peak t<sup>R</sup> 16.1 min corresponded to FAME 18:2 with the main fragments *m/z* 194.0 (α n-12) and *m/z* 248.1 (ω n-13), Figure 10A. The MBR value of 93 indicated two cumulated double bonds. It was interpreted as FAME 18:2n-12,13, most probably laballenic acid, highly abundant in *M. vulgare* seeds [61]. The fragmentation spectrum of FAME 18:2n-12,13 with the allenic system differed conspicuously from other arrangements of double bonds. The α fragment was accompanied by an α + 1 fragment with almost the same intensity, providing a double peak *m/z* 194/195 (Figure 10A). Analogous fragmentation behavior was also observed for other FAMEs with cumulated double bonds and helped us interpret allenic motifs in FAMEs. For instance, the compound eluting in 18.9 min was interpreted as FAME 19:2n-12,13. Its MS/MS spectrum provided *m/z* 208.1 (α n-12), *m/z* 209.0 (α n-12 + 1), and *m/z* 248.2 (ω n-13), corresponding to an MBR of 93 (Figure 10B). Analogously, peak t<sup>R</sup> 22.6 min showing *m/z* 222.1 (α n-12), *m/z* 223.1 (α n-12 + 1), and *m/z* 248.2 (ω n-13) was consistent with 20:2n-12,13 (spectrum not shown).

**Figure 9.** HPLC/APCI-MS base-peak chromatogram of FAMEs obtained from *Marrubium vulgare* seeds and the list of identified species.

**Figure 10.** APCI MS/MS spectra of the [M + 55]+• adducts of allenic FAMEs from *Marrubium vulgare* seeds interpreted as FAME 18:2n-12,13 (**A**) and 19:2n-12,13 (**B**).

In addition to allenic species, *M. vulgare* seeds contained FAMEs with conjugated double bonds. For example, the chromatographic peak t<sup>R</sup> 14.6 min represented FAME 18:2n-11,13. Its structure was deduced using *m/z* 182.1 (α n-13), *m/z* 208.1 (α n-11), *m/z* 222.1 (ω n-11), and *m/z* 248.1 (ω n-13), an MBR value of 107 (Figure 11A). Similarly, peak t<sup>R</sup> 17.4 min corresponded to FAME 18:2n-12,14 (Figure 11B). Overall, sixteen unsaturated FAMEs were detected in *M. vulgare* seeds, including monounsaturated, diunsaturated with allenic and conjugated double bonds, and triunsaturated species with methylene-interrupted double bonds (Table 4).

**Figure 11.** APCI MS/MS spectra of the [M + 55]+• adducts of conjugated FAMEs from *Marrubium vulgare* seeds interpreted as FAME 18:2n-11,13 (**A**) and 18:2n-12,14 (**B**).


**Table 4.** FAMEs identified in TG fraction of *Marrubium vulgare* seed lipids.

\* Tentative identifications.

#### 2.3.4. FAMEs from *Santalum album* Seeds

Indian sandalwood (*Santalum album*) is a tropical tree native to southern India and Southeast Asia. The oil from its seeds and seeds of other Santalaceae species is a rich source of acetylenic FAs. [96]. FAMEs obtained by the transesterification of the TGs from *Santalum album* seeds provided chromatogram shown in Figure 12.

The most abundant peak t<sup>R</sup> 10.3 min corresponded to FAME with 18 carbons and either three double bonds or a double and a triple bond. The MS/MS spectrum (Figure 13A) revealed the latter possibility, i.e., an acetylenic acid methyl ester. Diagnostic fragment *m/z* 190.1 and its satellite ion *m/z* 205.1 indicated a triple bond in the n-9 position (ω n-9TB). The corresponding α fragment (α n-9TB) at *m/z* 236.1 was not accompanied by a significant satellite ion at *m/z* 251.1, likely because of the triple bond conjugation with the n-7 double

bond. The α fragment *m/z* 262.0 and its satellite *m/z* 276.1 indicated a double bond in the position n-7. Low-intensity fragment ω n-7 was detected at *m/z* 166.1. The MBR value of 105 corresponds to a conjugated system of one double and one triple bond. The compound was identified as FAME 18:2n-7,9TB, most probably santalbic acid methyl ester.

˂

**Figure 12.** HPLC/APCI-MS base-peak chromatogram of FAMEs obtained from *Santalum album* seeds and the list of identified species. The inset shows a chromatogram of the same sample injected in 10× less amount.

ω

**Figure 13.** APCI MS/MS spectra of the [M + 55]+• adducts of acetylenic FAME from *Santalum album* seeds interpreted as FAME 18:2n-7,9TB (**A**) and 18:2n-7 TB,9TB (**B**).

α

α ω

ω

The MS/MS spectrum of a peak in 8.6 min revealed another acetylenic FAME with two triple bonds (Figure 13B). The ω fragment *m/z* 188.1 and its satellite peak *m/z* 203.1 indicated the triple bond at the position n-9TB, and the α fragment *m/z* 260.1 and its satellite *m/z* 275.0 the triple bond in n-7TB. The complementary α (n-9TB) and ω (n-7TB) fragments *m/z* 236.1 and *m/z* 164.1, respectively, were of low abundance. The MBR value calculated from the most abundant fragments (*m/z* 188.1 and *m/z* 260.1) equaled 103 and was consistent with two conjugated triple bonds. The compound was identified as FAME 18:2n-7TB,9TB .

The seeds oil was also found to contain acetylenic acids (FAMEs 18:3n-9TB, 20:2n-7,9TB) and conjugated acids (FAMEs 18:2n-7,9, 20:2n-9,11) not reported previously for *S. album*. In summary, FAMEs with triple bonds conjugated with either double or triple bond were found, together with saturated, monounsaturated, diunsaturated conjugated and methylene interrupted, triunsaturated, and tetraunsaturated species (Table 5).


**Table 5.** FAMEs identified in TG fraction of *S. album* seed lipids.

\* Composition of sandalwood oil ethyl esters reported in ref. [97].

#### **3. Experimental**

#### *3.1. Chemicals and Materials*

MS-grade acetonitrile and methanol (Sigma-Aldrich, St. Louis, MO, USA) were used as received. Chloroform, hexane, and diethyl ether were distilled from analytical-grade solvents (Penta, Czech Republic). Other chemicals, NaCl (≥99%, Sigma-Aldrich, St. Louis, MO, USA), di-tert-butyl-4-methylphenol (Fluka, Buchs, Switzerland), Rhodamine 6G (Sigma-Aldrich, St. Louis, MO, USA), and Diazald (99%, Sigma-Aldrich, St. Louis, MO, USA) were used. The standards of crepenynic acid (99%) and punicic acid methyl ester (purity 98%) were from Larodan (Malmö, Sweden), and 9-octadecynoic acid methyl ester, 9(*E*),11(*E*)-octadecadienoic acid methyl ester, and 9(*Z*),11(*Z*)-octadecadienoic acid methyl ester (all 98%) were purchased from Cayman Europe (Tallinn, Estonia). The standards were dissolved in chloroform at 1 mg/mL concentrations and stored at −25 ◦C. *Bombus pratorum* males were collected in the Czech Republic during the spring season and immobilization at −18 ◦C. Cold-pressed pomegranate seed oil (organic, unrefined) was from Biopurus Ltd. (Ashford, England). Seeds of *Marrubium vulgare* and *Santalum album* were purchased from a local garden center.

#### *3.2. Extraction and Transesterification of Lipids*

The samples were treated with organic solvents to obtain total lipid extracts. Briefly, peripheral fat bodies of three *B. pratorum* males were dissected and extracted with CHCl3/CH3OH (1:1, *v*/*v*) containing di-tert-butyl-4-methylphenol at a concentration of 25 mg/mL (500 µL

each) and sonicated for 15 min. The extract was collected using a Pasteur pipette. *M. vulgare* seeds (approx. 240 pieces; 0.25 g) or *S. album* seeds (5 pieces; 0.94 g) were crushed and extracted in methanol/chloroform (2:1 *v*/*v*, 10 mL) for 30 min. After filtration, 5 mL of 0.9% NaCl was added, shaken for few seconds, and the aqueous (upper) phase was removed. The cleaning step was repeated three more times with 2 mL of 0.9% NaCl solution.

Total lipid extracts or seed oil were separated by semipreparative TLC to isolate TGs. Pre-cleaned, in-house made silica-gel glass TLC plates (60 mm × 76 mm) and hexane/diethyl ether (80:20, by vol.) mobile phase were used. TLC zones were made visible by spraying Rhodamine 6G solution (0.05% in ethanol). A zone corresponding to TGs (*B. pratorum* R<sup>f</sup> = 0.36–0.55, pomegranate R<sup>f</sup> = 0.20–0.55, *M. vulgare* R<sup>f</sup> = 0.33–0.55, *S. album* R<sup>f</sup> = 0.30–0.55) was scraped off the plate and extracted with 10 mL freshly distilled diethyl ether. The solvent was evaporated to dryness under a nitrogen stream.

While TGs from *B. pratorum*, pomegranate seed oil, and *M. vulgare* seeds were transesterified in acidic conditions [98], base-catalyzed transesterification [99] was required for *S. album* lipids containing triple bonds. FA standards were methylated by diazomethane (synthesized in-house from Diazald). Diazomethane in diethyl ether was added dropwise to the FA solution in chloroform (10 mg/mL) until the color of the reaction mixture turned light-yellow. Unreacted diazomethane was deactivated by formic acid.

#### *3.3. RP-HPLC/APCI-MS and APCI-MS*

The liquid chromatograph consisted of a Rheos Allegro UHPLC pump, Accela autosampler with an integrated column oven, and an LCQ Fleet ion-trap mass spectrometer; the system was controlled by Xcalibur software (all Thermo Fisher Scientific, San Jose, CA, USA). Develosil RP-Aqueous C30 (250 × 4.6 mm, particle size: 5 µm; Nomura Chemical, Seto, Japan) stainless-steel column and isocratic elution with acetonitrile at 0.7 mL/min flow rate [20] were used. The chromatography proceeded at laboratory temperature except for *B. pratorum* sample separated at 40 ◦C. The injected volume of samples (standards and biological samples, 1 mg/mL and 10–20 mg/mL, respectively) was 10–20 µL. The APCI vaporizer and heated capillary temperatures were set to 380 ◦C and 180 ◦C, respectively; the corona discharge current was 2 µA. Nitrogen served both as the sheath and auxiliary gas at a flow rate of 50 and 20 arbitrary units, respectively. The MS spectra of positively charged ions were recorded in the *m/z* 180–470 range. The CID MS<sup>2</sup> spectra of [M + 55]+• were collected using a data-dependent analysis with an isolation width of 1.7 Da and normalized collision energy of 28%. The *m/z* range of MS<sup>2</sup> spectra was set automatically, depending on the precursor ion mass. The masses of the acetonitrile adducts for fragmentation were calculated as higher partners of the base peaks (*m/z* [M + H]<sup>+</sup> + 54 Da). The retention times and relative peak areas were obtained from ion chromatograms extracted for [M + H]<sup>+</sup> . The high-resolution MS data were recorded using an LTQ Orbitrap XL hybrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with an APCI ion source operated at the same conditions as for low resolution. The Orbitrap spectra were acquired at a resolution of 100,000 FWHM.

The standard (1 mg/mL) solutions were also analyzed by direct infusion to the mobile phase flow using the same APCI-MS conditions, as described above.

#### *3.4. Fragment Ion Abbreviations and Nomenclature*

The diagnostic ions in the MS/MS spectra of [M + 55]+• were denoted "α" if they carried the ester moiety or "ω" if they contained the terminal-carbon end without the ester group. The double bond position was indicated as α n-x and ω n-x, where x is the distance from the terminal end of the hydrocarbon chain. A triple bond was marked by "TB" in superscript.

#### **4. Conclusions**

This work demonstrates the applicability of acetonitrile gas-phase chemistry in APCI for characterizing the structure of polyunsaturated FAMEs. The reaction of C3H5N+• with

double and triple bonds occurs in the ion source, and the reaction products are fragmented to generate diagnostic ions. The method is highly versatile and suitable to many (if not all) arrangements of double and triple bonds in mono- and polyunsaturated chains. It was successfully applied to FAMEs with isolated, cumulated, and conjugated double bonds, triple bonds, and their combinations. The localization of the isolated double and triple bond positions is straightforward because of intense α and ω fragments. Distinguishing a double bond from a triple bond is easy based on the satellite fragments. While the satellite ions appear at +14 Da in the lipids with a double bond, they are found as intense +15 Da fragments in the case of a triple bond. When two or more unsaturated bonds exist in a chain, the spectra predominantly show α and ω fragments related to cleavages of C–C bonds before and after the unsaturated region. This can be utilized for deducing a possible arrangement of unsaturated bonds. A parameter named multiple bond region (MBR) can be calculated using the most abundant fragments and compared to tabulated theoretical values. The type and position of the unsaturated bonds within the unsaturated region can then be inspected in detail after focusing on less intense diagnostic fragments and their satellites. In the case of allenic FAMEs, the α fragment was accompanied by an intense α + 1 fragment, which gave a hint for the cumulated double bonds. When a triple bond was present in a polyunsaturated chain, it manifested itself by the +15 Da satellite peak accompanying the corresponding diagnostic fragment.

The localization of unsaturated bonds by HPLC/APCI-MS/MS with an acetonitrile mobile phase is a simple and convenient method. Since the derivatization occurs in the ion source during ionization, there is no need to perform the chemical modification of the analytes as a separate step before the analysis. Nominal mass resolution spectra were successfully used for the structure elucidation. However, high-resolution MS/MS data could help distinguish α and ω fragments, thus making the interpretation even easier. In this work, unsaturated FAMEs were characterized in *Bombus pratorum*, *Punicum granatum*, *Marrubium vulgare,* and *Santalum album*. The method's power is illustrated by the fact that, in addition to the known lipids, several new FAMEs were discovered. Although the method can also be applied to complex lipids [19,21,22], spectra interpretation is easier for lipids having only one fatty acyl chain.

**Supplementary Materials:** The following are available online, Figure S1: APCI MS/MS CID spectrum of the [M + 55]+• adduct of ricinenic acid methyl ester (FAME 18:2n-7*c*,9*c*); MBR = 107; Table S1: High-resolution data for fragments from APCI MS/MS spectra of FAME standards. Figure S2: APCI MS/MS spectra of the [M + 55]+• adducts of selected conjugated FAMEs from PSO interpreted as FAME 18:3n-5,7,9 (A), 18:3n-4,6,8 (B), 18:3n-2,4,6 (C), 18:3n-5,7,9 (D).

**Author Contributions:** Conceptualization, J.C.; methodology, V.V. and J.C.; investigation, P.H. and V.V.; resources, J.K.; writing—original draft preparation, P.H.; writing—review and editing, K.S.-P., P.H. and J.C.; supervision, J.C.; funding acquisition, P.H. and J.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Charles University Grant Agency, project number. 10119, the Charles University in Prague, project SVV 260560, and the European Regional Development Fund, OP RDE, No. CZ.02.1.01/0.0/0.0/16\_019/0000729.

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors wish to thank Stancho Stanchev for the preparation of diazomethane.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Sample Availability:** Samples of the compounds are not available from the authors.

#### **References**

