Round 1

Reviewer 1 Report

This work evaluated the cannabinoid content of THC and CBD in hemp (Cannabis sativa L.) using NIRS technology in combination with chemometric. The research content is creative and practical, but there are still some problems that need to be modified:

1. NIRS in the abstract needs to be supplemented with its full name.
2. Lines 109-112 need to be revised from Spanish to English.
3. The detection method and results of HPLC need to be more detailed.

Author Response

Overall comments

This work evaluated the cannabinoid content of THC and CBD in hemp (Cannabis sativa L.) using NIRS technology in combination with chemometric. The research content is creative and practical, but there are still some problems that need to be modified.

Response: Thanks for the time given for the corrections in the manuscript. We have been as through and detailed as possible in accomplishing with your suggestions. We appreciate your work, and we hope the manuscript is now acceptable for publication. Also, we have reviewed the paper making small modifications all over it, in order to improve its quality. Modifications are signaled in red typesetting.

Point 1: NIRS in the abstract needs to be supplemented with its full name.

Response 1: Thank you for your revision. As you suggested the full name of NIRS has been included in the abstract (lines 18-20): “The present work evaluates the cannabinoid content in hemp (Cannabis sativa L.) using Near Infrared Spectroscopy (NIRS) technology in combination with chemometric techniques”.

Point 2: Lines 109-112 need to be revised from Spanish to English.

Response 2: Thank you for your suggestion. We have eliminated these lines as it was a mistake.

Point 3: The detection method and results of HPLC need to be more detailed.

Response 3: Thank you for your comment. Considering your suggestion we have detailed the information related to the HPLC method and results as (lines 139-146 and lines 206-216): “The extraction was performed by ultrasound with methanol-chloroform subsequent decarboxylation. The mobile phase was acetonitrile (water (8:2 v/v), isocratic, stop time 8 min.) according to ‘Recommended methods for the identification and analysis of cannabis and cannabis products’ by United Nations Office on Drugs and Crime [19]. As to the result, for a qualitative identification, the retention time as well as the DAD spectrum of the cannabinoid have to match. The calculation for the quantitative results is carried out at the wavelengths of 220 and 240 nm.”

“For each of the samples analyzed, an individualized report was received with the sample identification data, the characteristics of the analysis methods used and the results obtained from the HPLC-DAD analysis. The results provided by the reference laboratory, expressed as a percentage, included data for humidity, THC total, CBD total, and 12 other cannabinoids that are not the subject of this study. 32 samples out of 35 were analyzed by HPLC-DAD, since 3 hemp plants infected with fungi were identified. The mean, the maximum, the minimum and the standard deviation of the reference results can be seen in Table 1.

Regarding the percentage of humidity, all samples presented values between 9.02 and 12.34 %. These values are between the ranges established in the delegated regulation (EU) 2017/1155 of the commission of February 15 of 2017 [33], which indicates a humidity between 8-13% in the preparation of cannabis samples.”

Reviewer 2 Report

This is well written and interesting paper in which the cannabinoid content in hemp using simple and chip NIRS technology combined with chemometric methods was evaluated. As reference method HPLC-DAD was used as reference total THC and CBD measurements. The obtained results show that the simple NIRS technology has a good potential for quantitative determination of cannabinoids.

I have only two comment:

• The manuscript should be thoroughly verified. See verses 109-112, which are in Spanish!!!
• Also the discussion of the results and the conclusions should be improved and need more comments with comparison of results from other papers dealing with cannabinoid determination presented in the literature.

In conclusion, the manuscript needs to be improved, before assessment about its suitability for publication in Agronomy is made. 

Author Response

Overall comments

This is well written and interesting paper in which the cannabinoid content in hemp using simple and chip NIRS technology combined with chemometric methods was evaluated. As reference method HPLC-DAD was used as reference total THC and CBD measurements. The obtained results show that the simple NIRS technology has a good potential for quantitative determination of cannabinoids.

Response: Thanks for the time given for the corrections in the manuscript. We have been as through and detailed as possible in accomplishing with your suggestions. We appreciate your work, and we hope the manuscript is now acceptable for publication. Also, we have reviewed the paper making small modifications all over it, in order to improve its quality. Modifications are signaled in red typesetting.

Point 1: The manuscript should be thoroughly verified. See verses 109-112, which are in Spanish!!!

Response 1: Thank you for your suggestion. We have eliminated these lines as it was a mistake.

Point 2: Also the discussion of the results and the conclusions should be improved and need more comments with comparison of results from other papers dealing with cannabinoid determination presented in the literature.

Response 2: Thanks for your suggestion. We have rewritten the discussion section enriching it with more and substantial references (lines 333-381) as: “Interest in the determination of the cannabinoid content of Cannabis sativa has in-creased in recent years, as indicated by the various studies published in this field. As in this study, other authors have achieved good results for the prediction of THC and CBD by NIRS without any data pretreatment. Thus, Sánchez-Carnerero et al. [6] obtained an R²cv of 0.99 for CBD and an R²cv of 0.99 for THC with an FT-NIR instrument. However, using a NIR Systems 6500 scanning monochromator for CBD prediction a SNV-DT pretreatment was applied obtaining an R²cv of 0.99 and a MSC for THC with an R²cv 0.98 and an RPD of 3.07.

In a more recent study, Deidda et al. [38] explore the feasibility of using NIRS for the quantitative analysis of THC. For this study, two handheld NIR spectrophotometers were used and compared, a low-cost device (NIR-S-G1) and a mid-cost device (MicroNIR onsite W 1700). Both entire inflorescence and resin samples were analyzed, and the reference method used was UHPLC coupled to UV detection. A preliminary study was conducted on 26 entire inflorescences that were then ground and sieved in order to evaluate the impact of sample homogeneity on the THC content predictions. Researchers obtained a THC concentration far wider than in our study, ranging from 0.92 to 22.21%. RPD values between 1 and 4.54 were obtained for the different physical forms of samples using both devices. In general, the MicroNIR spectrophotometer outperformed NIR-S-G1. More-over, 45 resin samples were analyzed with both devices obtaining an RPD value of 2.26 with the MicroNIR and 1.51 with NIR-S-G1. Therefore, authors concluded that the mid-cost system was best-suited spectrophotometer for their application.

In another study carried out by Chen et al [39], a different approach was adopted where the authors explore the potential of NIRS for the in-situ determination of CBD in hemp oil. For their study, 20 hemp oil samples with different concentrations of CBD and CBDA (determined by HPLC) were analyzed by a Bruker MATRIX-F FT-NIR spectrometer covering the 4000 to 12 000 cm^-1 range. Super partial least-squares regression (sPLSR) and self-optimizing support vector elastic net (SOSVEN) were applied to predict the concentrations of CBD achieving promising results with a coefficient of determination for the validation set > 0.98 and an RMSEV of 6.4 ± 0.1 mg/mL.

Besides, as mentioned in the introduction, spectroscopic techniques have been used to differentiate fiber-type from drug-type Cannabis sativa L. [40]. In their study, attenuated total reflectance - Fourier’s transform infrared (ATR-FTIR) in the 5000−400 cm⁻¹ region was used to assess 36 samples of C. sativa inflorescences, 8 of them were of drug-type, 14 of fiber-type and another set of 14 were cannabis samples at low THC concentration. PLS models were developed to predict the content of 7 seven neutral and acidic cannabinoids (THC, THCA, CBD, CBDA, CBG, CBGA, CBN). Authors achieved very good results with an R²cv higher than 0.99 for each cannabinoid and RMSECV values ranging from 0.020 to 0.163.

Also, mid-infrared (MIR) spectroscopy has been explored for the prediction of THC and CBD content in C. sativa. Thus, Geskovski et al. [41] used an ATR Fourier transform infrared spectrometer, in the 1700 to 400 cm-1 range, to quantify the content of THC and CBD in 45 flowers and 34 Cannabis extracts. PLS models were developed for both type of samples obtaining good results with and R²p and RMSEP values of 0.99 and 2.32% for THC, 0.99 and 1.33% for CBD, respectively for the flower samples and an R²p and RMSEP values of 0.95 and 3.79% for THC, 0.99 and 1.44% for CBD in the Cannabis extract samples, respectively.

In the current study, we present a feasible and low-cost method for the THC and CBD content determination in C. sativa samples using NIRS. Promising results were obtained for both cannabinoids indicating the potential of NIR technology as a predictive tool.”

Also, we have rewritten the conclusion as (lines 383-406): “The functionality of NIRS for the quantification of THC and CBD as principal canna-binoids in hemp (Cannabis sativa L.) along with their related spectral peaks was evaluated in this study. According to the latter, the region of the NIR spectrum analyzed presents characteristic absorption bands around 1210 nm, 1450 nm, 1736 nm, 1762 nm, 1820 nm, 1940 nm, 2060 nm and 2090 nm that are typical of proteins, lipids, water and other com-pounds present in hemp (from OH, NH, CH and other bonds). In addition, the band at 1736 nm, related to aromatic hydrocarbons of the terpenes, was associated with the CBD content, since cannabinoids are terpene-phenolic compounds.

Moreover, predictive models of the cannabinoid content in hemp have been obtained combining NIR spectroscopy and chemometric analysis. The best results for the prediction of both THC and CBD were obtained using the raw data, providing a simpler form of analysis. For the THC, the best PLS model achieved a determination coefficient of cross-validation of 0.77 and an RPD value > 2 which indicates its predictive capacity. For the CBD, the best PLS model achieved a coefficient of 0.77 and an RPD value > 2 also indicating the goodness of the prediction model.

Although the number of samples in this study was limited due to the high cost of HPLC, it has allowed us to demonstrate the potential of NIRS for the determination of main cannabinoids content in samples of Kompolti variety. Due to the goodness of the models and the results obtained, this study could be extended including a larger number of samples or other varieties of industrial hemp with a wider concentration of THC.

The results obtained here demonstrate that NIR spectroscopy offers speed and simplicity unmatched by other traditional techniques and accordingly, it has been tested as an alternative method to conventional HPLC analysis for the evaluation of cannabinoid content with promising results.”

Point 3: In conclusion, the manuscript needs to be improved, before assessment about its suitability for publication in Agronomy is made.

Response 3: Thank you again for your valuable review. We hope that with the changes made throughout the paper it is now suitable for publication in Agronomy.

Reviewer 3 Report

The manuscript titled Potential of NIRS Technology for the Determination of Cannabinoid Content in Industrial Hemp (Cannabis sativa L.) is interesting, well written and the methods are appropriate. However I found some mistakes:

Lines 109-112: please change the language and write in English.

Table 1: under THC total (%) , please change second Min. Should be Max.

Line 227: (figure 1) should be (Figure 1)

Author Response

Overall comments

The manuscript titled Potential of NIRS Technology for the Determination of Cannabinoid Content in Industrial Hemp (Cannabis sativa L.) is interesting, well written and the methods are appropriate.

Response: Thanks for the time given for the corrections in the manuscript. We have been as through and detailed as possible in accomplishing with your suggestions. We appreciate your work, and we hope the manuscript is now acceptable for publication. Also, we have reviewed the paper making small modifications all over it, in order to improve its quality. Modifications are signaled in red typesetting.

Point 1: Lines 109-112: please change the language and write in English.

Response 1: Thank you for your suggestion. We have eliminated these lines as it was a mistake.

Point 2: Table 1: under THC total (%), please change second Min. Should be Max.

Response 2: Thanks for your suggestion. It was a typo that has been corrected.

Point 3: Line 227: (figure 1) should be (Figure 1)

Response 3: Thank you for your comment. We have corrected the typo (line 230) as suggested.