1. Introduction
Bovine milk contains 3–5% of fat, making it one of the major components of milk solids alongside lactose and proteins. Milk fat is present as microscopic globules with triacylglycerols (TAG) as the core content and tri-layer phospholipids as the globule membrane. While TAG is considered mainly an energy source, phospholipids including mainly phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelin (SM), phosphatidylserine (PS) and phosphatidylinositol (PI) are bioactive molecules, offering multiple benefits to human health [
1,
2,
3]. As a result, there is a growing interest in using milk fat globule membrane material as an ingredient of infant formulas [
4]. In addition, some phospholipids of milk were found to be potential biomarkers for the health status of dairy cows [
5]. Consequently, accurate quantification of phospholipids in milk, isolated milk fat globule membrane and other dairy products is gaining increasing attention [
6,
7].
Several analytical systems have been reported for milk phospholipid measurement, the most common ones being LC-ELSD, shotgun-MS, LC-MS and
31P-NMR. Normal-phase LC-ELSD/Hilic-ELSD has been used for milk phospholipid analysis for over two decades (for review: see Liu et al. [
8]). It provides separation of the major phospholipid classes of milk (PC, PE, SM, PS and PI), and the ELSD response is thought to be independent of fatty acid (FA) structures of the lipids [
9]. This is a clear advantage over an MS detector. However, the drawbacks of this detector include: (1) no species information can be obtained; (2) it is prone to interference by co-eluting analytes as ELSD is a universal detector; (3) it has lower sensitivity as compared to MS.
Several studies have been reported recently on the use of
31P-NMR for phospholipid quantification in milk and dairy products [
10,
11,
12]. Although the sample preparation for NMR is simple and quantification of phospholipids based on
31P signal is reliable, the intrinsic low sensitivity of NMR remains the main obstacle for its widespread use. Despite the perceived shortcomings of shotgun-MS (especially that of ion suppression for low-abundance lipid species), the direct-infuse-MS method has been proven to be valuable for high throughput lipid profiling and is being frequently used in lipidomic analysis [
13,
14,
15,
16,
17,
18,
19].
The most widely used technique in lipidomic analysis of biological samples including milk is indisputably LC-MS (for reviews: see [
8,
20,
21]. For example, RP-LC-MS has been a popular choice in global lipidomic profiling of human plasma of large cohorts, enabling several hundred lipid species (polar and non-polar) to be quantified in a single analysis [
22]. Hilic-MS is also suitable for phospholipid analysis and has been applied to milk phospholipid profiling in numerous studies [
23,
24,
25,
26,
27]. Like normal-phase-LC, Hilic separates different polar lipid classes based on their head group but provides minimal separation of species within each class.
For both RP-LC-MS and Hilic-MS, the variation of ESI-MS response with FA structure is the major challenge for lipid quantification in milk and other biological samples [
18,
21,
28], due to the huge number of lipid species present in milk and very few lipid standards being commercially available. Using Hilic-QqQ-MS, Fong et al. [
29] demonstrated that the MS response is mainly determined by the head group whereas the FA structure has little influence when the concentration of phospholipids is below a certain level. This finding is significant and is a strong justification for Hilic-MS-based polar lipid quantification using one standard for each class [
26,
27,
30]. Despite being not fully validated, this one-standard quantification approach has also been widely adopted in RP-LC-MS-based polar lipid and TAG analysis [
31,
32,
33,
34]. This has led to the commercialisation of some proprietary lipidomic standard mix containing one deuterated standard per lipid class by Avanti Lipids. Although reliable for relative quantification (or fold-change comparison between sample groups), the utility of using one single standard for absolute quantification of lipids by RP-LC-MS remains unclear.
When using ESI-MS, the ionization efficiency (or response) of lipids is expected to be influenced by mobile phase composition, additives and sample matrix. While mobile phase composition at a specific retention time (RT) is determined by the elution gradient and the most frequently used mobile phase additives include ammonium formate and ammonium acetate, the sample matrix is in turn related to the lipid extraction methods (e.g., Folch method vs. one-phase method). How each of these factors influences the lipid quantification results remains to be investigated.
Compared to small molecules encountered in metabolomics study, lipids are complex molecules containing a large number of carbon and hydrogen atoms, so the contribution and interference of C13 isotope in particular is expected to be significant for both identification and quantification [
35]. However, until now, few researchers have considered this in lipidomic analysis [
18,
19,
36,
37]. Clearly, both type 1 isotope (monoisotopic proportion) and type 2 isotope (mainly M+2) corrections are relevant to milk lipid quantification, but whether both corrections are necessary for the quantification of phospholipids and TAG remains to be clarified.
In this study, we have systematically evaluated several key factors including lipid extraction method, LC stationary phase, mobile phase buffer, LC elution gradient, MS resolution settings as well as type 1 and type 2 isotope corrections for their effect on milk phospholipid quantification at the species and class levels. The five major phospholipid classes (PC, PE, SM, PS and PI) were measured in this study.
4. Discussion
Quantification of lipids especially phospholipids in milk and dairy products are gaining increasing interest for their perceived beneficial effects on human health. While normal-phase-LC coupled to ELSD detector is the classic method for class-level phospholipid quantification, 31P-NMR is gaining popularity for its simplicity in sample preparation and reliability in quantification. However, for species-level quantification, LC-MS is still the preferred option for most researchers.
While numerous studies have been devoted to comprehensive identification of milk lipid molecular species, accurate quantification of lipids at the species level is currently unachievable. Three reasons are responsible for this. (1) A large number of isomer species have been identified in milk. (2) Chromatographic separation of all these isomers is currently unfeasible regardless of LC column type, length and elution gradient. Indeed, a multi-dimensional chromatography system may be needed to achieve this goal. (3) The number of lipid standards is very limited as compared to the thousands of species identified in milk. Consequently, until now lipid quantification with milk samples has been mostly performed at the sum structure level (i.e., the sum of all isomers having the same molecular mass and the same chemical formula) for both TAG and polar lipids.
Numerous workflows have been reported for lipidomic analysis of biological samples in the past 20 years. The large number of options concerning lipid extraction, LC separation, MS detection and quantification methods are overwhelming and a standard or consensus workflow for milk lipid quantification is lacking. To address this, we undertook a systematic evaluation of the current procedures for quantification of the major lipid classes in bovine milk.
The Folch method is still the most popular protocol for lipid extraction from milk, although the one-phase method we reported several years ago is much simpler [
39]. In this study the one-phase method was found to be associated with a slight ion enhancement for SM and PS regardless of LC methods. It is known that the matrix derived from one-phase extraction is more complex than the Folch matrix, but the underlying mechanisms for the ion enhancement remain to be investigated. Overall, the one-phase method is as suitable as the Folch method for lipid quantification either by RP-LC-MS or by Hilic-MS. In addition, the higher efficiency of the one-phase method in extracting PS and PI from milk (as compared to the Folch method) has been confirmed. As a result, the application of the one-phase method is encouraged for LC-MS-based milk lipidomic analysis, especially for the processing of large number of samples.
Regarding the mobile phase additives, both ammonium formate and ammonium aetate have been widely used in lipidomic analysis workflows [
41,
42]. Our study revealed that the former offers a much higher MS response for most lipid classes, hence a better sensitivity than the latter. Although the underlying mechanism for the differential ionization efficiency of lipids in the presence of these ammonium salts is unknown, this is the first report showing the advantage of using ammonium formate as mobile phase buffer in lipid analysis by LC-MS.
Quantification of lipids is often carried out with one standard per lipid class due to the limited availability of standards [
27,
43]. Although either the one-point IS calibration method or one-standard external calibration method is useful for relative comparison or for determination of fold-change between sample groups, the reliability of such approaches for accurate quantification of lipids at the species- or class-level requires a thorough investigation.
Like C18, C8 provide species-level separation, facilitating lipid identification. However, such a separation also means that different species elute at different retention times, some close to while others distant from the IS. The ionization efficiency of phospholipids is primarily determined by the head group. However, retention times, which are related to mobile phase composition, could also affect the ionization of analytes. Consequently, the response factors are expected to be different for different species of the same lipid class when using RP-LC-MS. In such a case, the accuracy of quantification using one single standard is questionable. The fact that inconsistent results were obtained with two RP-LC elution programmes is a direct proof for this. To compensate for the response variation across the spectrum of a lipid class, multiple standards were used in the quantification of SM species [
19].
Adding IS prior to lipid extraction is the best way to correct any errors related to extraction efficiency, solvent evaporation, inject volume variation and detection fluctuation. Unfortunately, in the case of milk lipids, only deuterated standards are suitable to be spiked into the samples, as endogenous lipids are present for any molecular mass. Given the high cost of deuterated lipid standards, adding them to each sample will significantly increase the analytical cost. Through the post-extraction IS spiking test, we have found the matrix effect is modest when the milk matrices are appropriately diluted, suggesting that the external calibration method (with deuterated or non-labelled standards) can be used for quantification to reduce the sample processing cost. In such a case, the signal variation caused by the injection volume inaccuracy and/or detector fluctuation can be offset by performing replicate analyses, given the extraction efficiency by the Folch and the one-phase method has been well proven.
For polar lipid analysis, in addition to RP-LC-MS, Hilic-MS is widely used [
8]. Although the coelution of all species within the same class hinders the identification caused by M+2 isotope interference, the overall peak shape is clearly better than RP-LC and the quantification results are more consistent regardless of elution gradients. Owing to its robustness and high compatibility with ESI-MS, Hilic-MS has become a popular technique for polar lipid analysis of milk.
The type 1 and/or type 2 isotope corrections are rarely applied in milk lipid analysis. We have shown in this study that type 1 correction may be necessary when using one or a few standards for each lipid class. As for the type 2 correction, it is essential for Hilic-MS-based lipidomic analysis unless a high-resolution mass analyser is used, as the mass difference of M+2 isotope of one species with the mono-isotope of another one is about 0.009 Da. According to Bielow et al. [
15], a mass analyser with a resolving power of around 170,000 is required to resolve such a mass distance for ions with
m/
z around 760. When used at a resolution of 240,000, Orbitrap Elite can achieve an actual resolution between 170,000 and 175,000 at
m/
z 760, thus allowing suitable separation of M+2 isotopes of phospholipid species of milk. Alternatively, a lower-resolution MS (≤60,000) is unable to resolve M+2 isotope from the adjacent species, which could lead to overestimation of several-fold of the latter depending on the relative abundance of the pair if type 2 correction is not performed.
When RP-LC- and Hilic-MS are compared for quantifying phospholipids of the same milk samples, a large difference was observed for the relative proportion of lipid species as well as the sum concentration of PC, SM and PE classes, highlighting the complexity of accurate quantification of lipids using LC-MS. Based on our findings, for phospholipids, we recommend a workflow combining one-phase extraction with Hilic separation, one-point IS calibration and a high-resolution MS (or low-resolution MS plus type 2 isotope correction).
In conclusion, various key steps in lipid quantification protocols have been compared in this study. While the reliability of the one-phase method for lipid extraction from milk has been confirmed, a significant variation in lipid quantification results was observed between RP-LC-MS and Hilic-MS and even between RP-LC elution programmes. In addition, we have demonstrated the importance of type 1 and type 2 isotopic corrections in lipid quantification. Our study highlights the urgent need for a comprehensive LC-MS and 31P-NMR cross-validation for milk phospholipid quantification and calls for the production and commercialisation of reference milk (or milk powder) samples and a deuterated lipid standard mix suitable for milk lipid analysis.