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Development and validation of a High Performance Liquid

Chromatography-Tandem Mass Spectrometry method for the
absolute analysis of 17 α D-amino acids in cooked meals

Cecilia Barbas-Bernardos , Isabel Garcia-Perez ,

Maria Paz Lorenzo , Vanesa Alonso-Herranz , Jeremy Nicholson ,
Antonia Garcia

PII:
DOI: https://doi.org/10.1016/j.chroma.2019.460598
Reference: CHROMA 460598
S0021-9673(19)31006-4
To appear in: Journal of Chromatography A
Please cite this article as: Cecilia Barbas-Bernardos , Isabel
Received date: 14 June 2019 Garcia-Perez , Maria Paz
Revised date: 25 September 2019 Lorenzo ,
Accepted date: 3 October 2019 Vanesa Alonso-Herranz , Jeremy
Nicholson , Antonia Garcia ,
Development and validation of a High Performance Liquid Chromatography-Tandem Mass
Spectrometry method for the absolute analysis of 17 α D-amino acids in cooked meals, Journal of
Chromatography A (2019), doi:
https://doi.org/10.1016/j.chroma.2019.460598

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Highlights:
• Optimization of an LC-MS/MS method for absolute quantitation of 17 D-AAs in food
• Validation of the method with cooked meals and LLOQ are 2.0 ng/mL for most of them
• Application of the method to healthy breakfast, lunch and dinner meal samples  Absolute
levels of D-AAs are given per daily food intake and within each main meal.
• Method applicable to quality control purposes in food
Development and validation of a High Performance Liquid Chromatography-Tandem Mass Spectrometry
method for the absolute analysis of 17 α D-amino acids in cooked meals.

Authors:

Cecilia Barbas-Bernardos*

Isabel Garcia-Perez# Maria

Paz Lorenzo*

Vanesa Alonso-Herranz*

Jeremy NicholsonƗ

Antonia Garcia*

*Center for Metabolomics and Bioanalysis (CEMBIO), Facultad de Farmacia, Universidad CEU San Pablo,
Boadilla del Monte, ES-28668, Madrid, Spain; cec.barbas.ce@ceindo.ceu.es (C.B.-B.); antogar@ceu.es
(A.G.).

#Section of Biomolecular Medicine, Division of Integrative Systems Medicine and Digestive Diseases,
Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, London, UK.

ƗAustralian National Phenome Center, Health Future Institute, Murdoch University South Street, Perth WA
6150, Australia.

Corresponding author:
Antonia García (Corresponding author):
Center for Metabolomics and Bioanalysis (CEMBIO)
Facultad de Farmacia
Universidad CEU San Pablo
Campus Monteprincipe
Boadilla del Monte
28668 Madrid, Spain
E-mail: antogar@ceu.es
Tel.: +34 913724753; fax: +34 913724712

Keywords:
Chiral derivatizing agent

D-Amino acids

Enantioseparation

LC-MS/MS

Food samples

Cooked meals
ABSTRACT

In the nutrition field, there is a lack of understanding about the impact that dietary chiral composition may
have on health, especially regarding cooked meals. Chiral amino acids (AAs) are naturally present in food
and their proportion may vary quite a lot. Besides, the D-amino acids (D-AAs) are present in very low
concentration compared to L-AAs, so very sensitive methods are required for their accurate quantitation.
Moreover, some of them have been described as indicators of quality and different food processes. In this
research, we propose a robust method for the absolute quantitation and enantiomeric ratio of 17 D-AAs in
cooked meals. The AAs were extracted from 1 g of the homogenised meal with methanol, derivatised with
(S)-N-(4-nitrophenoxycarbonyl) phenylalanine methoxyethyl ester ((S)-NIFE) and analysed by RP-LC-
MS/MS. The separation was carried out with an Acquity BEH C18 (100 mm x 2.1 mm, 1.7 µm) column at
70º C, with 10 mmol/L ammonium bicarbonate in water as eluent A and acetonitrile as eluent B at a 0.3
mL/min flow rate in gradient elution. The MS operated in positive electrospray ionisation method in
multiple reaction monitoring (MRM) mode. Isotopically labelled AAs were used as internal standards for
the quantitation. The method was validated for 17 D-AAs in the cooked food samples in terms of
specificity, linearity, precision, accuracy, matrix effect and stability. LLOQ are 2.0 ng/mL for most of them.
Additionally, linearity was also studied for L-AAs. After optimization and validation, the method was
applied to real breakfast, lunch and dinner samples of cooked meals (n=18) that were part of a diet with a
very high concordance with WHO dietary guidelines. Level of concentration of major and minor D-AAs
have been described per total daily intake and within each of the three main meals. This method can be
used for quality control purposes as well as to investigate the role of chiral composition in food and clinical
outcomes.

Acknowledgments:

Funding: This work was supported by the Spanish Ministry of Science, Innovation and Universities MICINN
RTI2018-095166-B-I00 C. B.-B. thanks for the financial support provided by the Spanish Ministry of
Education, Culture and Sport (FPU 15/03032) and (EST16/00831)

Conflict of interest: The authors declare that they have no competing interests.

1. INTRODUCTION:
Lifestyle has drastically changed in the past century and with it the dietary habits of large populations.
People in developed countries are exposed to fast food, mass production of dietary components and wider
importation of different types of foods. This changing dietary landscape has a direct impact on the risk of
developing non communicable diseases (NCD) with the highest rate of morbidity and mortality worldwide
such as, diabetes, cardiovascular diseases and some cancers, as discussed in several clinical and
epidemiological studies [1-3].

During the last decade, research has focused on investigating the role of biologically active compounds in
certain foods to reduce the risk of developing NCD [3, 4], for the prevention of chronic diseases and for
maintaining good health [3]. However, it has been demonstrated the importance of studying dietary
patterns rather than individually look at nutrients in specific food [5]. On the contrary, there is a lack of
understanding of the impact that dietary chiral composition may have on health. Indeed, very little is
known about chiral composition, especially of cooked meals. Among all the compounds present in food,
chiral compounds are in high concentrations and their proportion may vary significantly. Amino acids
(AAs), one example of chiral compounds, are naturally present and they have an important nutritional
value [6]. D-Amino acids (D-AAs) are present in very low concentration regarding to their corresponding L-
AAs and have been described as indicators of different food production. Therefore, within the food
industry, chiral analysis has been used to study quality, age, processing and storage [7], asses adulteration
[8] and contamination [9], quantify pesticides, monitor fermentation [10], and to study the relationship
between food components and health [3, 11].

The presence of D-AAs in humans is due to the intake of food and beverages (raw and processed) and from
products of bacterial metabolism in the intestine. Around the 80% of the D-AAs are absorbed in the
intestine and metabolised in the liver and kidney by the enzyme D amino acid oxidase [12]. A low activity
of this enzyme may lead to the accumulation of these compounds, a condition that can have toxic effects.
D-AA isomers are increasingly being recognised as important signalling molecules and variations in their
levels in the organism have been described as marker of different pathologies. D-Phe was found increased
in a gestational diabetes mellitus study [13] and D-Phe along with D-Tyr were detected in higher amount in
patients with chronic renal failure [14]. In the case of the presence of DSer and D-Asp in the central
nervous system, it is known that they are products of the enantioconversion by different enzymes [15].
Others, such as D-Glu and D-Ala, have also been associated with the N-methyl-D-aspartate (NMDA)
receptors directly relating them with synaptic, learning and memory processes as
well as neural diseases [16, 17]. Ever since, the interest in D-AAs has increased in
many different research fields such as clinical, forensic, plant studies and food
industry. In the nutrition field, it is important to know the absolute amount as well as
the enantiomer ratio in food. Very little is known about their bioavailability
and their biological activity which are both dependent on, the source, the cooking
methods, the metabolic interactions and the diet and health status of the
consumer [18]. Hence, there is a need to precisely and accurately analyse their content in food.

Due to the latest advances in separation and detection techniques, the enantioseparation and
quantification methods have improved in terms of accuracy, precision and sensitivity. Since the presence
of the D-AA isomer in most of the samples is much lower than the L isomer, reliable quantitation of D-AA is
always a challenge. The measurement of D and L isomers can be performed by combining separation
techniques to different detectors as briefly described below.
The most common separation techniques for the study of D- and L-AAs are gas chromatography (GC), high
performance liquid chromatography (HPLC) and capillary electrophoresis (CE). The first two, with two
approaches [19]: (i) indirect methods based on the formation of diastereoisomers through the reaction
between a chiral derivatizing agent (CDA) with the chiral compounds present in the samples, followed by
the separation of the reaction product in a non-chiral stationary phase. Visser et al. compared seven CDAs
for the enantioseparation of AAs in urine, plasma and cerebrospinal fluid samples [15]. Among all, (S)-N-(4-
nitrophenoxycarbonyl) phenylalanine methoxyethyl ester ((S)-NIFE) demonstrated the best sensitivity,
enantiomeric purity, good stereoselective properties and mild derivatization conditions [15]. Five years
later, Xing Y et al. adapted this method for the analysis of 16 DAAs in plasma samples from rats with
Alzheimer´s disease [17]. (ii) Direct methods, based on the different interactions of the enantiomers with a
chiral stationary phase (CSP) or with a chiral selector that is added to the mobile phase, to separate the
product of the reaction, in a non-chiral stationary phase. In CE, the addition of a chiral selector to the
background electrolyte may enhance the separation of chiral compounds. In the last years, different
reviews about this topic have been published elsewhere [7, 20, 21].

These three separation techniques may be found coupled to different detection systems, mainly mass
spectrometry (MS), ultraviolet or fluorescence detectors (FD). Although Phe, Trp and Tyr are aromatic
compounds which can directly be analysed with absorbance or fluorescence ultraviolet-visible detection,
the sensitivity is not good enough and to enhance it, AAs are usually derivatised. Various derivatizing
agents have been used for absorbance detection; 2,4-dinitrofluorobenzene (DNFB), 9-fluorenylmethyl
chloroformate (FMOC-Cl), benzoyl chloride and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC)
[21] among others. Similarly happens with fluorescence detection, some examples of labelling agents are
the AQC mentioned before, o-phthaldialdehyde (OPA), 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F),
1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester) difluoroboradiaza-s-indacene (TMBB-Su) [21].
It is important to select those derivatives with excitation and emission wavelengths compatible with the
FD system. The main drawback of those detectors is that retention/migration time is the unique criteria for
the identification of each enantiomer and the purity of each single peak could be compromised in complex
samples.

MS increases the capability of the detection due to the selectivity of the mass spectra as additional
information. For MS analysis, several researches have been published both with and without CDA. In the
case of indirect approaches, with GC-MS, silylation agent such as N,O-
Bis(trimethylsilyl)trifluoroacetamide (BSTFA) [22] or perfluorinated anhydrides [13] were used to obtain
volatile derivatives prior to their separation in a CSP and (+)-α-methoxy-α-trifluoromethyl-phenylacetyl
chloride to obtain diastereomeric amides [23]. In LC, it is common to use (R)-1-boc-2-piperidine carbonyl
chloride [24]; ortho-phthalaldehyde (OPA)/ N-isobutyryl-L-cysteine (IBLC) reagent [25]; (R)-1-phenylethyl
isothiocyanate ((R)-AMBI) [15], 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marfey´s reagent) [26, 27];
2,3,4,6-tetra-O-acetyl-beta-d-glucopyranosyl isothiocyanate (GITC); (S)-NIFE; 4-(3-
isothiocyanatopyrrolidin-1-yl)-7-nitrobenzofurazan (NBD-PyNCS) etc. [15, 21] or homemade reactant as
Biaryl Axially chiral derivatizing agent [28]. There are two critical points to be considered when working
with CDA, their enantiomeric purity and the control of the racemization of the derivatives over the
chemical reaction. In the experimental comparison, among the list of seven derivatizing agents for the
quantification of D-AAs in biological samples, (S)-NIFE offers very good sensitivity from the MS-based
detection in positive ion mode and lack of racemization [15] and besides, it is commercially available.

Regarding direct approaches, in GC-MS mainly two types of CSP have been used, those based on chiral
amino acid moiety, such as Chirasil-L-Val (L-Val-tert-butylamide), or chiral cyclodextrin (cyclic
oligosaccharides), such as Rt-Dexsa column ((2,3-di-acetoxy-6-O-tert-butyl-dimethylsilyl gamma
cyclodextrin) [20]. With respect to LC-MS, many new chiral columns have been developed over the last
decades varying the chiral selector, always deriving from few classes of compounds. Most of the chiral
selectors come from macrocyclic antibiotics, carbohydrates, AAs (native or branched), proteins and
cyclodextrins [29, 30]. More recently chiral crown ethers stationary phases, such as CROWNPAK CR-I (+)
column have been successfully applied for AA enantioseparations in plasma or serum samples [31, 32].
There are some examples of CSP used to analyse biological samples and food samples [7, 33, 34]. Lately,
two-dimensional high-performance liquid chromatographic systems where a CSP along with a non-chiral
stationary phase is been used [35, 36]. In some cases, even though a chiral column was selected, chemical
derivatization was performed [37] mainly for enhancing substantially the detection sensitivity and the
peak symmetry due to the small size and the high polarity of the native AAs.

In short, chemical derivatization for indirect chiral separation has gained popularity in the recent years,
enabling the use of common reversed-phase columns, avoiding the complex coupling of two columns in
comparison to direct strategies. Also, the extraordinary capabilities of the MS detection allowed a
decreased of the limit of detection (LOD) and the limit of quantification (LOQ) compared to fluorescent
tags [20]. Among all, (S)-NIFE has demonstrated to be one of the best options due to (i) the absence of
racemization, (ii) short reaction time and (iii) high sensitivity of the MS-based detection in positive ion
mode.

Here we have optimised and validated a method for the analysis of D and L ɑ-AAs focused especially on
absolute quantitation of D-AAs in cooked food samples. The use of (S)-NIFE as CDA labelling agent
combined with LC-MS/MS with positive detection and isotopic dilution was selected after in depth analysis
of previous methods. After optimization and validation, the method was applied to real breakfast, lunch
and dinner samples of cooked meals that were part of a diet with very high concordance with World
Health Organisation (WHO) dietary guidelines [38]. To our knowledge, it is the first time that a chiral
analysis separating 17 pairs of enantiomers is applied to cooked meal samples in order to quantify the D-
AAs real intake in absolute and relative concentration.

2. MATERIALS AND METHODS

2.1. Samples:

Food samples, used to develop and optimise the sample treatment and the analytical method, were
collected during a randomised, controlled, crossover trial described elsewhere [38]. These foods are
duplicates of the meals provided to participants and are used as a proof of concept
for this project. The diet was designed to be the most concordant with WHO
healthy eating guidelines (Table S1) [38]. For the diet, there were three meals,
samples of cooked food corresponding to breakfast, lunch and dinner and cooked
individually (n=6) in six different weeks (Table S2). This cooked food
samples were blended, individually, by adding 250 mL of water to improve the
homogenization and kept at -80 ºC until the day of the analysis. A pool
sample (QC) for the optimization and validation of the method was prepared by mixing aliquots of meals
from all the samples of the study.

2.2. Reagents:

All standards, chemicals and reagents used, were of analytical grade. Isotopically labelled standards were
obtained from Cambridge Isotopes Laboratories (Andover, MA, USA) DL-Alanine [2,3,3,3-D 4, 9798%]; L-
Arginine:HCl [GUANIDO-15N2, 98%+]; L-Asparagine:H2O [15N2, 98%]; DL-Aspartic acid [2,3,3-D3, 98%]; DL-
Cysteine [3,3-D2, 98%]; DL-Glutamic acid [2,4,4-D3, 98%]; L-Glutamine [2,3,3,4,4-D5, 97%]; DLLeucine [D10,
98%]; L-Lysine:2HCl [4,4,5,5-D4, 96-98%]; DL-Methionine [3,3,4,4-D4, 98%]; L-Ornithine:HCl [3,3,4,4,5,5-D6,
98%]; DL-Phenylalanine [RING-D5, 98%]; DL-Proline [2,3,3,4,4,5,5-D7, 97-98%]; DL-Serine [2,3,3-D3, 98%]; L-
Threonine [13C4, 97-99%; 15N, 97-99%]; L-Tryptophan [INDOLE-D5, 98%]; L-Tyrosine [3,3-D2, 98%]; DL-Valine
[D8, 98%]) and (S)-NIFE, labelling grade, was from Santa Cruz Biotechnology (Heidelberg, Germany). The
non-labelled D- or L-AAs (purity > 98%) were purchased from Sigma-Aldrich (Steinheim, Germany).
Dilutions of standards, solutions and aqueous mobile phases were prepared with ultrapure water obtained
from a MilliQ® system (Millipore, Billerica, MA, USA) or with Methanol (MeOH) LC-MS quality from Sigma-
Aldrich (Steinheim, Germany). Formic acid (FA), ammonium bicarbonate, acetonitrile (ACN), trichloroacetic
acid 99%, 5-sulphosalicylic acid dehydrated >99% and HCl were also from Sigma-Aldrich (Steinheim,
Germany). Borate and sodium hydroxide (NaOH) both of analysis grade were from Panreac Quimica
(Barcelona, Spain).

2.3. Working solutions and standards for method validation:

Individual stock solutions of amino acid standards, labelled (AA*) and non-labelled ones (AA) were
prepared either in MeOH or ultrapure water or their binary mixture, with a concentration of 1,000 µg/mL
(ppm), aliquoted and kept in the freezer (-20 ºC). A 20 µg/mL intermediate individual labelled standard
solution was prepared by dilution and from it a working standard solution with a mixture containing 200
ng/mL (ppb) (L enantiomers) and 400 ng/mL (D+L enantiomers). For the different levels of the calibration
curves, intermediate and working solutions of non-labelled standards were prepared by mixing them
accordingly. Several sample blanks were used along the method development and at the beginning and at
the end of the sample analysis [39]. All stock solutions were defrosted and diluted the day of analysis.

2.4. Sample extraction protocol

One gram of homogenised food, accurately measured, was mixed with 8.0 mL of MeOH in a screw glass
tube, gently vortexed for 1 min and let stand 15 min at room temperature to enable the extraction of AAs
from the food. Samples were centrifuged at 15 ºC (4,000 g for 15 min).

2.5. Derivatization protocol for standard solutions and samples

30 µL of supernatant of the extracted samples or 30 µL of the working standard solutions were placed in a
safe-lock polypropylene tube (1.5 mL) for the derivatization step and 30 µL of the internal standard (IS)
solution (containing 200 ng/mL (ppb) of each D- and L- labelled AA) was added. To concentrate the
sample, the supernatant was evaporated to dryness using a SpeedVac Concentrator System (60 ºC for 30
min). The residue was resuspended in 50 µL of water and 35 µL of 0.15 mol/L sodium tetraborate solution
(pH=9.0) and the mixture was mixed for 10 min. For the derivatization, 50 µL of (S)-NIFE solution in ACN
(15 mg/mL) were added, vortex-mixed for 15 s and incubated at room temperature for 20 min. The
reaction was stopped by the addition of 15 µL of 4 mol/L cold HCl and vortex-mixed for 15 s again. The
polypropylene tubes were left at -20 ºC for 30 min to enhance salt precipitation, and then ultracentrifuged
at 4 ºC (15000 g for 10 min) and the supernatant was transferred to the LC vials (Figure
1).
Figure 1

2.6. LC-MS analysis:

We conducted the analysis on an LC-QqQ-MS system consisted of a degasser (1260 Infinity series, Agilent),
a binary pump and an autosampler (1260 Infinity II series, Agilent). Autosampler was kept at 12°C during
the analysis. The chromatographic separation was performed at 70ºC on a reverse-phase column, Acquity
UPLC BEH C18 column (100 mm x 2.1 mm, 1.7 µm) with an Acquity UPLC BEH C18 precolumn (5 mm x 2.1
mm, 1.7 µm VanGuard Pre-Column) from Waters (Mildford, MA, USA). The mobile phase consisted on 10
mmol/L ammonium bicarbonate in water pH=9.5 (eluent A) and ACN (eluent B) pumped at 0.3 mL/min
with a total run time of 37.5 min. A post-gradient with the following proportions (v/v) of solvent B was
applied: 0-4.8 min at 8% B, 4.8-10.8 min 8% to 20% B, 10.8-12.6 min 20% to 22% B, 12.6-14.4 min 22% B,
14.4-18.3 min 22% to 47% B, 18.3-21.0 min 47% B, 21.0-24.0 min
47% to 50% B, 24.0-25.0 min 50% B, 25.0-26.0 min 50% to 55% B, 26.0-27.0 min 55% B, 27.0-28.0 min 55%
to 95% B, 28.0-32.0 min 95% B, 32.0-33.0 min 95% to 8% B and 33.0-37.5 min 8% B (equilibration time).
The injection volume was 5 μL. Data was collected in dynamic multiple reaction monitoring (MRM) mode,
with an electrospray ionization source (ESI) in positive ion method, in a Triple Quadrupole analyser
(G6470A, Agilent). The capillary voltage was 4000 V, with a scan rate of 500 scans per second. The
nebulizer was set to 45 psi, the gas temperature 350 ºC and the flow rate was 10 L/min. The sheath gas
temperature and flow rate were 400 ºC and 10 L/min respectively. All the ions, MRM condition and
retention times are included in Table S3. Quantification was performed by the stable isotope dilution
method. (Area Amino Acid (AA)/Area labelled AA (AA*)) considering the same D- or L- AA* (labelled
standard). When the corresponding D-AA* was not available, its counterpart L-AA* was selected. For D-
and L- His, the peak area of D-Ser* and L-Ser* was used respectively, and for D-Ile and L-Ile the peak area
of D-Leu* and L-Leu* was used.

2.7. Method validation

According to US Food and Drug Administration (FDA) document for bioanalytical method validation, the
method was evaluated with respect to specificity, linearity, low limit of quantification (LLOQ), precision,
accuracy, matrix effect and stability. The method was validated by the stable isotope dilution method
(Area AA/Area AA*) with labelled AA (AA*) for the analysis of the D and L enantiomers of 17 α- AAs by the
LC-MS-QqQ platform in cooked food samples. Labelled IS were selected to allow considering matrix
effects, i.e. determination of concentration correctly, despite presence of co-eluting matrix components.
As labelled IS, the same D- or L-AA* was selected. When the corresponding D-AA* was not available, its
counterpart L-AA* was selected. For D- and L-His, the peak area of D-Ser* and L-Ser* were used
respectively, and for D-Ile and L-Ile the peak area of D-Leu* and L-Leu* were used.

Specificity was evaluated by comparing the MRM chromatograms of (S)-NIFE

derivatised i) D- and L-AAs ii) D- and L- labelled internal standards and iii) the pool of
food sample.

Linearity for D-AAs was tested by analysing, in triplicate, a mixture of D-AAs in

MeOH over the concentration range of 2.0 to 1,000 ng/mL (ppb). Six levels of
concentration were used covering from the LLOQ to 1,000 ng/mL (ppb), always at least 500% of the mean
values obtained in preliminary analysis performed with a pool of samples. Each level contained 200 ng/mL
of each enantiomer in the mixture of labelled internal standards. Linearity for L-AAs was performed
independently by analysing in triplicate six levels of concentrations covering a concentration range of 0.20-
10.0 µg/mL (ppm) of a mixture of L-AAs in MeOH and 200 ng/mL of each labelled internal standard.

The LOQ was estimated by the signal-to-noise ratio (SNR=10). To determine the LOQ for each AA, a
standard containing each AA at 2.0 ng/mL concentration was analyzed. The LOQ was calculated as follows:
LOD = 2.0 ng/mL × 10/SNR. and LLOQ was experimentally checked with the lowest concentration level of
the calibration curve for precision and accuracy.
To prove the constancy of the instrumental response to each analyte, instrumental precision was tested
with 10 injections of the D-AA solution in the mid-range of the calibration curve.

Once the standard solutions with the D- or L- AAs mixture were prepared, all were processed as described
previously (derivatization protocol for standard solutions and samples).

Linearity, precision and quantitation of samples were performed by the internal standard calibration
method considering the relative response of the D-AAs or L-AAs in the sample respect to the
corresponding D- or L-labelled AAs.

Intra and inter-day precision of the method: Identical samples of QC were derivatized independently and
analysed at four concentrations across the assay range, including LLOQ, low, mid and high QCs in the same
day (n=3) and in two different days respectively (n=6).

Accuracy was assayed in triplicate, by comparing the areas of spiked samples. These were prepared by
mixing extracted sample with D-AAs in a linear range including low, mid and high QCs (from 20 to 250
ng/mL and LLOQ both intra-day (n=3 each) and inter-day (n=6 each level).

Precision was expressed as the CVs (% RSD), and accuracy was expressed as a relative error.

Matrix effect
Matrix effects on analyte quantification were assessed by comparing the slope of the linear regression
curves in the linearity with standards (n=18) and the slope of the linearity with samples from the accuracy
study (n=15).
%ME =100*(1-(Slope matrix/Slope standards)

With this equation, positive results mean signal suppression. Additionally, ME% values between -20% and
+20% are within the accepted ranges [40].

Stability

Based on previous experience with chiral analysis of D- and L-AAs [13], stock solutions should be kept in
the freezer and defrosted just once the day of the analysis. Stability of individual stock solution of labelled
AAs and post-preparative stability of samples were studied. Considering their high price, stability of
labelled AA stock solutions was studied after one year at -20 ºC. Moreover, post-preparative stability was
carried out by analysing derivatised real pool of sample for 0, 10, 24, 36, 48 and 60 h under 12 ºC in the
autosampler tray.

2.8. Quantitation of the D-AA in real samples

The quantitation of D-AAs and L-AA (µmol/g) in the food samples of the study, was based on their relative
area respect to the same D-AA* or L-AA* internal standard and multiplying by the accurate concentration
of the IS (200 ng/mL) according to the equation 1. MW: molecular weight. Wsample: weight of food sample in
g.

Equation 1: C (µmol/g): AreaAA*CIS (ng/mL) *8 /(AreaIS*1000 * MW * Wsample)

Some of the D-AAs* were not available, and the labelled standards were present just as the L-AA*
enantiomer. For them, relative response factors RRF of D-AAs respect to L-AAs were calculated by dividing
the Area of the D-AA by the Area of the L-AA obtained with a 200 ng/mL (ppb) solution with both D and L-
AAs by triplicate. The quantitation of D-AAs (µmol/g) in the food samples of the study, was based on the
equation 2. RRFD-AA: relative response factor. MW: molecular weight. Wsample: weight of food sample in g.
Equation 2: C (µmol/g): AreaD-AA*CIS (ng/mL) *8 /(AreaIS(L-AA)* RRFD-AA*1000 * MW * Wsample)

2.9. Data treatment

System control and data analysis were performed with Agilent Mass Hunter QQQ Qualitative and
Quantitative software programs (version B.07.00).

2.10. Statistical analysis

Following the data processing of the samples from the controlled study, uni and multivariate (UVDA and
MVDA) data analysis were performed. For the UVDA, non-parametric test (Mann–Whitney U or
Wilcoxon rank sum test) (p≤ 0.05) were performed to assay the statistically significant AAs in every
individual meal. Regarding the MVDA, SIMCA version 14 (Umetrics, Umea, Sweden) was used to obtain the
unsupervised Principal Component Analysis (PCA) plot.

3. RESULTS AND DISCUSSION

The sample treatment for cooked food samples, together with the chromatographic LC-MS/MS ESI+
method was optimised based on the extraction of AAs in a polar and volatile organic solvent, the reaction
of the AAs’ enantiomers with the CDA and then the resolution and detection of the enantiomers.

3.1. Sample extraction

The derivatization was optimised considering the low concentration of D-AAs respect to the L counterpart,
and the extraction of the AAs from the sample. Therefore, different extraction methods were assayed: (i)
MeOH (ii) Acidulated water: 1 mol/L FA in water (H2Oac) (iii) MeOH mixed with H2Oac (1:1) (iv) ACN (v)
Trichloroacetic acid in water 10% (w/v) and (vi) Sulphosalicylic acid in water 5% (w/v). In all cases, 8 mL
was the extraction volume used for 1 g of sample.

Even though AAs are polar compounds, water could not be used alone, because proteins must be
precipitated and removed. Otherwise, the column and the injector could be clogged. Both trials ii & iii
where H2Oac was used, solid particles were observed in the supernatant after
centrifugation.

Approaches (i) and (iv) gave clear supernatants. After trials (v) and (vi)
some solid particles appeared and were centrifuged twice to eliminate them through
the pellet. This procedure was not enough so, they were filtered with a 0.22 µm
nylon membrane to get rid of the solid particles.

Chromatograms from all the conditions, except for (ii) and (iii), were compared and the highest signal was
obtained with condition (i). Extracted ion chromatograms of most of AAs showed the highest intensities in
the case of pure MeOH, consequently this was the solvent selected for sample extraction (data not
shown). The final conditions are depicted in figure 1.
 3.2. Optimization of the derivatization conditions

The (S)-NIFE was chosen because its sensitivity and enantioselective properties had already been proved
by Visser et al. [15]. Derivatization requires basic pH [41] so, sodium tetraborate was the buffer added.
Moreover, pH>9 was confirmed.

First of all, following the recommendations of Visser, we tested the enantiopurity of the labelling agent by
reacting with a pure L-AA standard solution (L-Ala). After checking a single peak in the MRM
chromatogram (with the transition of the corresponding derivative), we started with the optimization of
the method.

Afterwards, different volumes of MeOH were tested (5, 8, 10, 12 mL) concluding that 8 mL was the most
adequate as a compromise between ion suppression and sensitivity (data not shown).
Finally, different volumes of supernatant were assayed keeping constant the amount of (S)-NIFE. Having
chosen MeOH as extractant, 15, 30 and 45 µL of supernatant were derivatised and analysed. The
increment in the signal from the three injections was not proportional to the increase in the sample
volume for the highest one. Then 30 µL was selected.

Based on previous literature, 12.5 mg/mL of (S)-NIFE in ACN were selected for food samples with 1224% of
proteins in order to assure enough excess of reagent for whole cooked samples and to promote the
synthesis of derivates.

3.3. Chromatographic optimization

For the separation of the whole set of D- and L-AAs simultaneously, in the positive ion mode, a basic
mobile phase is required. The mobile phase selected was the one used by Visser et al. [15]. Eluent A
contained 10 mmol/L ammonium bicarbonate with a pH=9.5, likewise in other works except for one,
where the concentration used was 8 mmol/L with no reference to its pH [17]. The flow used was adjusted
to 0.3 mL/min in order to not overpass the threshold of 600 bars corresponding to the HPLC pump.
Expectedly, the performance of ESI source with these lower flow rate could be better.

The mobile phase gradient was modified to reduce the total run time while increasing the enantiomeric
resolution. The resolution obtained for each pair of D/L AAs was higher than 1.5 (e 1) except for Ser and
Lys (1.3 and 0.93 respectively). Under the best conditions, D and L enantiomer of Glu and Asp were still not
baseline separated. In a very recent study 19 D-AAs were analysed using liquid chromatography coupled to
ion mobility-mass spectrometry (LC-IM-MS) in milk from different origins [42]. Chromatographically, they
also observed that the L enantiomer eluted before the D. In their study they did state the separation
between D- and L-Asp and D- and L-Glu. This might suggest that the third analytical dimension, based on
their drift time, improved the selectivity. Two peaks were detected for derivatised L-Phe and they were
able to prove that it came from the same analyte.

Table 1

Xing Y et al. were also able to enantioseparate D-Asp with UHPLC-MS/MS in rat plasma, though they did
not mention the use of isotopically labelled AAs [17]. Furthermore, the IS used was Fudosteine, a
compound eluting at the end of the chromatogram (instead of their labelled standards) without being
representative of the differences in the mobile phase composition by the gradient elution. Visser et al. had
already suggested different mobile phase to analyse the two acidic AAs, thereby complete separation of
Asp and Glu enantiomers was achieved with isocratic elution (0.1% (v/v) FA, 87% water and 12.9% ACN).
ESI performance is affected by the mobile phase composition, this means ionization is not equal along the
gradient. Therefore, the use of all the isotopically labelled D-AAs could enable a better quantification. In
the case of His and Ile, since their respective AA* could not be obtained, the IS with the closest retention
time in the chromatogram, Ser and Leu, were respectively selected for their quantification.

Finally, different MS/MS parameters were tested for the derivatised D-AAs. As stated in Visser et al. and
previously by Olajos et al., Cys, Lys and Tyr reacted with two molecules of (S)-NIFE. This was also observed
for Orn. Using Optimizer software (Agilent Technologies), the different data acquisition parameters for
MRM were optimised for each individual AA (Table S3). In all the cases, the product ion selected was m/z
120, yet other authors could have used different transitions. Xing Y. et al. targeted m/z 382.4 -> 133 as the
transition for Asp and the product ion selected for Arg was m/z 200.9 [17]. In the case of His, the product
ion selected was m/z 110.1 and so did Visser et al. Besides, there are several differences regarding the
product ions for Asp, Glu and Arg (m/z 134.1, m/z 148.1 and m/z 201.13 respectively) [15].

Regarding Phe, a very intense peak corresponding to m/z 415.1 (Phe-NIFE derivative) was observed during
preliminary trials with standards. Hence, the detection of such a big peak showed the seed of doubt
regarding the possibility of quantifying Phe. Considering the reaction scheme described elsewhere [41],
(S)-NIFE reacts through the amino group in the AAs, D or L, to generate the diastereomeric derivative and
4-nitrophenol in equimolar concentrations. Besides these, two side-products had been described [41], the
so called “symmetric urea”, N,N'-bis(3-phenylpropionic acid methoxyethyl ester 2yl)urea and the “Phe-
ester”, phenylalanine methoxyethyl ester. The presence of the last one was explained as spontaneous
decomposition of the carbamic acid. But it has never been described that, following this loss of CO 2, the
decomposition continues generating Phe alone. Since this newly produced Phe is in the same solution as
(S)-NIFE, they both react producing Phe-NIFE diastereomers. Figure 2 depicts this process. Figure 2.A
represents the MRM from d5-DL-Phe (420.0-> 120.0) in a mixture of DAAs standard solutions spiked with
IS. Figure 2.B represents the MRM transition of D-Phe (415.1->120.0) in a mixture containing non-labelled
D-AAs. Figure 2.C represents the transition of Phe in a blank containing only MeOH and (S)-NIFE. The blank
(2.C) generates only one peak, which coincides, in RT, with the peak of d 5-L-Phe. This peak is also observed,
with high intensity, in the mixture containing pure D enantiomers. This is explained because during the
reaction between an AA and (S)-NIFE, the L enantiomer of Phe is formed (due to (S)-NIFE decomposition),
meaning this method could only be able to quantify D-Phe-NIFE derivatives under these conditions.

Figure 2

DL- Orn has been enantioseparated, for the first time, expanding the number of
AAs that can be analysed using (S)-NIFE [15, 17, 42]. Moreover, the bis-derivative
was detected. In spite of the D-AAs being in much lower concentrations, the
baseline separation obtained was enough to selectively detect and quantify the D
isomer eluting after the L.

3.4. Method validation

Specificity

There was no significant endogenous interference coming from the reagent, IS, or sample in the
measurement of the D-AAs and L-AAs except for Glu and Asp (not enough resolution) and L-Phe
(sideproduct of the reagent). Figure 3 shows some examples regarding the specificity study comparing
sample, standards and labelled standards.
Figure 3
Linearity and low limit of quantification (LLOQ)

For linearity, the correlation coefficients (r) were all above 0.990 except for D-Phe (0.95) in which only
from 200-1000 ng/mL (ppb) were demonstrated to be linear but with high bias. The coefficients of the
linear regression along with their confidence interval and LOQ (SNR=10) as well as LLOQ, checked
experimentally, were listed in Table 2. Linearity was confirmed, and non-bias was found for all D-AAs with
only very little bias for D-Ile and D-Lys. LLOQ values were 2.0 ng/mL (ppb) for all D-AAs except for D-Tyr
(5.0 ppb), D-Lys (20 ppb), D-Cys (40 ppb), D-Phe (200 ppb). Peak area for D-Cys were very little for the
lowest concentrations of the linearity likely due to the instability of thiol group. For D-Phe, the response
for the lower concentrations resulted much higher than expected and increasing within the time,
unquestionably coming from the reagent, so it was definitively discarded for the next trials of the
validation study. Sensitivity regarding obtained LLOQ were in the range of very few ng/mL, like the most
sensitive published methods [43, 44].

Table 2

In the linearity study with L-AAs, the correlation coefficients (r) were all above 0.990. The coefficients of
the linear regression along with their confidence interval are listed in table 3. Linearity was confirmed, and
non-bias was found for all L-AA with very little bias for L-Arg, L-Asn, L-Ile, L-Leu, L-Pro, and L-Tyr. As the
goal of this research was the absolute and relative quantitation of D-AAs in cooked food samples, the
complete validation of L-AAs was not performed except the linearity study. Moreover, the quantitation of
L-AAs was performed by the stable isotope dilution method, with the corresponding LAAs* standards,
allowing also the relative quantitation as % D respect to the total AAs content.
Table 3

Precision and accuracy

Instrumental precision was evaluated by the RSD of 10 consecutives injections of the same standard
solution corresponding to the mid-level of concentration of the linearity study with D-AAs. RSD values
were in the range 0.67 to 2.4% (Table 4). The intra- and inter-day precision and accuracy of the method
were determined by the analysis of QC samples at the LLOQ and three different concentrations: low, mid
and high level. As shown in Table 4, the RSD of all the LLOQ were below 17% and the accuracy was in the
range 100 ± 20%. The RSD for the three QC samples were all less than 9.2% in the same day and the
accuracy ranged from 84.0% to 115.2% for D-AAs in cooked food. We can state from the consistency and
reproducible values depicted in Table 4 that all were acceptable.

Table 4

Matrix effect and stability

The matrix effect (ME) was calculated for 17 D-AAs as % (Table 4). The highest value corresponded to DLys
(14.3%), D-Orn (13.7%) and D-His (12.3%). The values for the rest of D-AAs were lower than 10% when
quantification of D-AAs was based on the stable isotope dilution method (Table 4).
The values in Table 5.A showed the stability of freshly prepared 200 ng/mL (ppb) from individual stock
labelled standard solution (1000 µg/mL) after 12 months in the freezer (-20 ºC). Racemization is
demonstrated in d6-L-Ornithine and in d8-DL-Valine because the D-AA* has increased up to 28.0% and
12.6% respectively. For the rest, the variation in the % of D-AAs* was less than 10%.
Table 5.B showed the results (in %D-AA) obtained in the stability study post derivatization with cooked
food samples at different times: 10, 24, 48 and 60 h. According to the values, after 24 h in the autosampler
at 12 ºC, most of D-AAs and L-AAs remained constant with very little variation (<7.0%). DGln and D-His
underwent the highest variation after 24h: 10.0% and 8.4% respectively. For L-AAs, variations were all
below 6.0% except L-His (8.5%). Even after 60h, variations of many D-AAs such as DAla, D-Arg, D-Ile, D-Leu,
D-Met, D-Orn, D-Pro, D-Ser, D-Thr, D-Trp, D-Tyr and D-Val as well as all L-AAs (except L-His) were lower
than 10.0%. The isotopic dilution of the samples with the labelled standards and the calculation by the
stable isotope dilution method facilitates these good results.

Table 5

Comparation with other LC-MS/MS methods

When comparing our method with other LC-MS methods, this is the first one developed to be applied for
absolute quantitation of D-AAs in cooked meals. Lately, several papers have been published describing
chromatographic or electrokinetic separation with pure standards, but very few with real applications [30]
Among them, Table 6 depicts representative validated methods, with several examples in bioanalysis
(plasma, serum, CSF, saliva or brain), but scarcely with food samples (little variety of samples). Recently,
just one study analyses native AA enantiomers with direct approaches (CSP) though it cannot be applied to
DL-Pro, L-Gln, and D-Lys [32]. Among the most sensitive methods, however, are those based on
derivatization with labelling agents.
Derivatization time in our method is 20 min, while derivatization reaction rates vary substantially with
other CDA, even with overnight derivatization. For example, 40 min, from 30 min to 24 h, or 3h are
required with (S)-2,5-dioxopyrrolidin-1-yl-1-(4,6-dimethoxy1,3,5-triazin-2-yl) pyrrolidine-2-carboxylate
(DMT-(S)-Pro-OSu) [45], with Marfey’s reagent or with (R)-1-Boc-2-piperidine carbonyl chloride
respectively; moreover the former is not commercially available. In addition, the coverage of AAs
enantiomers in our work is higher than others just focused on D-AA enantiomers. Besides, D-Orn was not
determined by any other method.

Even though the use of labelled internal standards can help to overcome the
quantification problems related to the sample matrix-induced effect, this
strategy is not always followed as described in Table 6. Regarding the elution order,
retention time should be lower for the D-AA than for the L-AA but that is the less
common scenario. The throughput of our LC-MS/MS method, with 33.0 min of
analysis time, is slightly lower than others based mainly on UHPLC-MS/MS but
much higher than the method based on 2D nano LC-MS/MS (515 min). Additionally, chromatographic
resolution along with the intra-day and inter-day precision are like others published by different groups
with LC-MS/MS. The most sensitive method for a set of D-AAs is the one based on two-dimensional nano
LC-MS/MS. The sensitivity of our method is comparable to the most sensitive LC-MS/MS methods,
according to the LOQ values depicted in the Table 6 with LOQ for D-AAs in the range of 0.075 to 27.8 pg
(amol range), especially for D-Iso, DPro and D-Orn (Table 6).

Table 6
Quantitation of the pool of food sample for the validation study

The pool of food sample for the optimization and validation of the method was analysed independently
(n=6) with the validated method and the results are depicted in table 7. Obtained results in the pool of
cooked food samples allow us, for the first time, to organise D-AAs in samples by their level of
concentration, being the most abundant in absolute terms: D-His and D-Ala (57.2 and 8.0 µmol/g
respectively) and the most abundant in percentage resulted to be D-His, D-Cys and D-Ser (26.7, 10.0% and
0.92% respectively) and the less abundant D-AAs are D-Tyr and D-Lys (not detected). Table 7

3.5. Application of the method to a study with real food samples

The proposed method was applied to cooked meal samples for the simultaneous quantification of 17 DAAs
in absolute and relative ratio. Results are based on the analysis by LC MS/MS of 18 samples (breakfast,
lunch and dinner samples) from one diet with very high concordance with WHO dietary guidelines. Each
individual sample was spiked with the mixture of labelled standards (200 ng/mL each) in order to perform
the absolute quantification of the D enantiomer by the relative area under de curve (AUC) respect to the
corresponding D labelled IS, according to the equation 1 and 2 described previously.

As far as we are concerned, this is the first time that cooked food meals are characterised in terms of its
chiral AA profile. In order to compare the similarities and differences of the 18 samples of the study, a non-
supervised multivariate analysis PCA was performed considering the concentration of free D-AAs and L-
AAs as well as the relative abundance of D-AAs with respect to the total amount of free AAs present in the
food (sum of the D- and the L-AAs abundancies) (Figure 4). As it can be seen in that figure 4, the chiral
composition of the meals was compared using four different angles. Firstly (Figure 4A) considering the
individual contribution of each AA enantiomer (D- and L-AA) in each meal: breakfast, lunch and dinner,
secondly considering only the concentration of D-AAs (Figure 4.B), thirdly considering only the
concentration of L-AAs, and finally considering the ratio of D-AAs (D-AA/ (D+L)-AA) of free AAs intake in
each food.

Figure 4

When representing all the models in figure 4 (A-D), the 6 meal breakfast clusters separate from the other
two meals by the first component while the second component differentiates between lunch and dinner.
This can be explained due to the similarities of lunch and dinner that are the same type of meal in
relationship to the breakfast meal [38]. The highest explained variance R 2 corresponds to models 4.C and
4.D, those in which the amount of the most abundant enantiomer AA and the less abundant respects to
the total concentration of the AAs are represented. Therefore, the dual chiral information could be the
best for classification of the food samples. Besides, breakfast contained many more fermented foods, a
process known to induce changes in the chirality of AAs, and that could be why the models based on the
either absolute or relative concentration of D-AAs (4.B and 4.D) show the highest dispersion in the
breakfast samples. On the other hand, lunch and dinner normally resemble a lot in how they are prepared
and the type of food they contain. Equally important is the dual chiral composition (absolute concentration
of D-AAs and D-AAs ratio) since low absolute concentrations could come from meals with lower
concentration of proteins.

Furthermore, we have investigated the amount of individual D-AAs consumed in one day and its
distribution into the three main meals, (Figure 5, 6, 7). The findings were organised by level of
concentration. So, the highest daily intake (Figure 5) corresponds to D-His and D-Ala (in the range of 160-
40 µmol/g of sample) and then D-Asn and D-Gln (in the range of 10 µmol/g of sample), D-Pro, D-Ser and D-
Trp are between 1-5 µmol/g, while the rest of D-AAs: D-Arg, D-Cys, D-Ile, D-Leu, D-Lys, D-Met, DOrn, D-Thr,
D-Tyr and D-Val are below 1 µmol/g. Moreover, some D-AAs were not detected neither in lunch nor in
dinner samples: D-Lys and D-Tyr.

A non-parametric statistical analysis Kruskal-Wallis and Wilcoxon rank sum test was performed to find
differences in the levels of individual D-AAs among breakfast, lunch and dinner samples (Figures 6 and 7).
Regarding absolute concentration (Figure 6), the highest differences were found while comparing
breakfast and lunch: D-Ala, D-Arg, D-Asn, D-Gln, D-His, D-Ile, D-Orn, D-Ser, D-Thr, D-Trp, D-Tyr and D-Val
were found statistically different. Moreover, no differences were found only for five D-AAs: D-Cys, DLeu, D-
Lys, D-Met and D-Pro. As previously detected by the PCA models, very few D-AAs were found statistical
different between lunch and dinner: D-Ala, D-Gln, D-His and D-Trp.
Figure 5

Figure 6

Considering the ratio of each D-AA (D-AA/(D+L)-AA) depicted in figure 7, the highest D-AAs ratio
corresponds to D-His (35-45% in any of the three meals) and D-Cys (around 10%). In the 1-5% D range we
found D-Ala, D-Asn, and D-Ser (2.5-5% in breakfast) and the other D-AAs are present in a ratio below 1%.
Among then, the lowest D-AAs ratio (<0.1%) corresponds to D-Arg, D-Gln, D-Thr, D-Trp, D-Tyr and DVal.
Additionally, fourteen D-AAs appeared to be statistically different in breakfast respect to lunch and dinner:
D-Ala (only vs dinner), D-Arg, D-Asn, D-Cys (only vs dinner), D-Gln, D-His, D-Leu, D-Met, D-Pro, DSer, D-Thr,
D-Trp, D-Tyr and D-Val, and all of them except D-His were found higher in breakfast.
Figure 7

Regarding L-AAs, figure 8, the most abundant are L-Gln and L-Ala (10 and 1 mmol/g respectively) and then
L-Lys, L-Thr and L-Val (0.5-1 mmol/g). Lunch is the meal with the highest level of L-AAs compared to
breakfast for all the L-AAs except L-Cys and L-Trp and also compared to dinner
except for L-Leu, L-Lys, LOrn, L-Ser and L-Trp.

Figure 8

Summing up, breakfast contains the highest absolute amount of D-AAs for D-Arg,
D-Asn, D- Pro, D-Ser, DTrp, D-Tyr and D-Val in absolute values and fourteen D-AAs
were found higher in relative concentration: D-Ala, D-Arg, D-Asn, D-Cys, D-Gln, D-His, D-Leu, D-Met, D-Pro,
D-Ser, D-Thr, D-Trp, D-Tyr and D-Val. Lunch contains the highest amount of D-AAs for D-Ala, D-His, D-Gln,
D-Orn and D-Thr in absolute values and only D-His in relative amounts. Besides, dinner contains the
highest concentration of D-Ile compare to breakfast and lunch, while none was found the highest in
relative concentration. This study provides a viable method for the selective and stereospecific absolute
and relative quantification of 17 D-AAs in cooked food and it offers new possibilities of studying their
biological value in nutrition and health.

Generally, the presence of D-AAs in food is due mainly to bacteria ubiquity (controlled or uncontrolled)
and food treatments (microwave, high temperature, alkaline media). Several processes where bacteria
participate could lead to an increase in the number of D-AAs. Some have been described as part of the
bacteria cell wall and bacteria cytoplasm (D-Ala, D-Met, D-Orn, D-Thr, D-Tyr, and D-Val) [46] and have
therefore been used to monitor bacterial contamination of foodstuffs [46, 47]. Foods undergoing
fermentation processes will also have higher amounts of D-AAs. Researches haven been published relating
their presence with lactic acid fermentation (D-Arg, D-Ala, D-Asn, D-Leu, D-Lys, D-Met and DSer) [48].
More specifically, fermented dairy products contain D-Ala, D-Lys, D-Pro, D-Thr and D-Val [33, 47-51];
kimchi has D-Ala, D-Ile, D-Met, D-Thr and D-Tyr [33, 50, 52] and vinegar contains also these as well as D-
Asn, D-Pro and D-Val [33, 47, 48, 50-52]. In wine, D-Lys, D-Tyr and D-Val have been detected [47, 53].
Apart from bacterial related processes, D-AAs have also been detected in fruit and vegetables juices and
beers containing these juices (D-Arg, D-Lys, D-Pro, D-Ser and D-Val) [47, 48, 51, 54], in honey (D-Lys and D-
Ser) [47], and D-Ala, D-Ile, D-Phe and D-Tyr have been quantified in cocoa powder [55].

Different food treatments have shown to convert the L forms into D as is the case of D-Ile and D-Pro in tea
during the fragmentation of the leaves [56]. Irradiated ham contained several D-AAs (D-Ala, D-Asp, D-Leu,
D-Ser, D-Val) [57] and microwave-treated formulae of infant food contained D-Pro [58]. Cooking, baking or
frying may induce racemisation too [46]. Some investigations performed by several authors indicate that
10 to 40% of the AAs content of feather meal produced by means of alkaline hydrolysis undergoes
racemisation as a function of the production parameters [46]. This conversion from the L-AAs to D-AAs
may alter the biological value and safety of food by (i) forming useless AAs, (ii) forming peptide bond non-
breakable by proteolytic enzymes and (iii) forming antagonists or nutritionally inactive AAs [6]. Moreover,
organoleptic properties of food vary with the enantiomeric ratio [58].

High amounts of D-AAs could be deleterious as the unique source of dietary protein [59]. In general, D-
AAs digestibility is lower and the nutritional value of the meals could be affected by their respective DAAs
level of concentration. D-Trp levels, in order to replace L-Trp functions’ related to growth, need to be 2.5
times higher [6]. D-Pro has been described as substrate of Helicobacter pylori, a bacterium related with
gastric inflammation and ulcers [60]. Therefore, its presence in food should be studied. Moreover, as far as
we are concerned, there has not previous information described about D-Cys, Gln and D-His in food.

This study has demonstrated the importance of combining both absolute and relative D-AAs amount in
cooked meals in order to compare the daily intake of D-AAs (breakfast and lunch and dinner) or just the
amount coming from each meal in absolute and relative values. Relevant finding based on this information
can be used for unveiling the role of D-AAs in health and for quality control purposes. Furthermore, this
methodology could enable the classification both of different meals/menus and different diets.

Eventually, we propose to use this methodology for quality control and for research of the effect of
variation regarding D-AAs either in absolute and relative concentrations in health.

4. CONCLUSIONS

In this paper, we propose a robust, optimised and validated method for the analysis of D-AAs in cooked
meals. The combination of the extraction of AAs with methanol, labelled with (S)-NIFE and RP-LC-MS/MS
has demonstrated to be a well suited CDA for the indirect chiral analysis of AAs in food. No racemization
takes places and the derivatised D- and L-AAs have a good stability and enantioselectivity for 17 AAs
enabling their simultaneous quantification by the stable isotope dilution method with labelled standards.
Time required for derivatization is 20 min and 38 samples can be analysed each day.

The developed method has enabled the characterisation of the D-AAs profile of a whole meal, and its
absolute and relative quantification, for the first time. Major and minor D-AAs have been described in
breakfast, lunch and dinner, along with the level of concentration and the total intake. This method can
give information about the enantiomer level of concentration in absolute and relative value and could be
used for quality control purposes as well as nutritional values, processing flavour and aroma including
several D-AAs without previous knowledge about them in food such as D-His and D-Gln.

This therefore suggests, the importance of investigating the impact that the chiral composition of food has
on health.
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FIGURE CAPTIONS
Figure 1. Scheme of the food sample treatment. First the extraction of the amino acids followed by the
derivatization reaction
Figure 2. MRM chromatograms of (S)-NIFE derivatised standards obtained by LC-MS/MS. D and L isomers
are indicated. A: transition of d5-DL-Phe (420.0-> 120.0) from a mixture of non-labelled D-AAs standard
solutions spiked with labelled IS; B: transition of D-Phe (415.1->120.0) in a mixture of standards containing
pure non-labelled D-Phe; C: blank of MeOH containing (S)-NIFE alone giving a peak with the transition
(415.1->120.0) in the RT of the D-Phe pure standard.
Figure 3. MRM chromatograms of (S)-NIFE derivatised standards obtained by HPLC-MS/MS. D and L

isomers are indicated in each case. Green: food sample; Orange; D-AAs standards at 40 ppb; Purple:
labelled standards at 200 ppb.

Figure 4. PCA-X score plots for the three meals together both in absolute values (A-C) and ratio (D). A: D-
and L- AAs included as independent variables; B: D-AAs; C: L-AAs; D: Ratio D-AAs/(D+L) AAs. Blue:
breakfast; yellow: lunch; brown: dinner. Models have an explained variance (R 2) of: A. R2=0.769; B.
R2=0.680; C. R2=0.885; D. R2=0.810. UV scaling.

Figure 5. Daily intake content of D-AAs (breakfast, lunch and dinner) in µmol/g.
Error bar: SEM

Figure 6. Absolute concentration of D-AAs (µmol/g) in breakfast, lunch and dinner

(n=6). Error bar: SEM. p<0.05. * Breakfast vs Dinner. # Breakfast vs Lunch. †
Lunch vs Dinner. NS: non-significant. ND: not detected.
Figure 7. Ratio of D-AAs (D-AA/(D-AA+L-AA)) in breakfast. lunch and dinner (n=6). Error bar: SEM.
p<0.05. * Breakfast vs Dinner. # Breakfast vs Lunch. † Lunch vs Dinner.
Figure 8. Absolute concentration of L-AAs (µmol/g) in breakfast, lunch and dinner (n=6). Error bar: SEM.
p<0.05. * Breakfast vs Dinner. # Breakfast vs Lunch. † Lunch vs Dinner.

TABLES:

Table 1. Resolution between D- and L-AAs measured in a cooked food sample during the validation.
*Measured in breakfast; ** measured in the d5-DL-Phe in a standard solution containing only MeOH and
IS.

Rs

RT (min)
L- D-
Ala 9.9 12.1 2.9
Arg 12.0 12.6 3.9*
Asn 7.3 8.5 2.5
Cys 20.6 20.8 3.1
Gln 8.3 9.7 5.0
His 8.7 10.5 1.7
Ile 15.0 16.8 4.3
Leu 15.4 17.2 5.1
Lys 20.3 20.5 0.93*
Met 14.1 15.5 4.1
Orn 20.1 20.4 2.0
Phe 16.6 18.5 11.0**
Pro 11.8 12.7 1.9
Ser 8.2 9.5 1.3
Thr 9.5 11.9 3.2
Trp 16.9 18.6 5.6
Tyr 21.1 21.4 2.0*
Val 13.3 15.2 6.0
Table 2:

The calibration parameters, LOQ (estimated) and LLOQ (checked) of 17 D-amino acids in the study with
real ready-to-use food. (A) Linear from 200-1,000 ng/mL
Linearity study Sensitivity

RT Range LOQ (pg) LLOQ

Slope Intercept r IS
(min) (ng/mL) (SNR=10) (ng/mL
D-Alanine 12.1 2.0–1000 67.5±2.2.10-4 -0.6±1.0.10-1 0.998 1.1 2.0 D-Ala*
D-Arginine 12.6 2.0–1000 50.9±1.8.10-3 -7.9±8.3.10-2 0.998 5.4 2.0 L-Arg*
D-Asparagine 8.5 2.0–1000 126.1±6.5.10-5 -1.7±3.0.10-2 0.995 2.8 2.0 L Asn*
D-Cysteine 20.8 40–1000 46.5±3.2.10-4 -1.7±2.1.10-1 0.994 27.8 40.0 D-Cys*
D-Glutamine 9.7 2.0–1000 218.3±7.5.10-5 -1.6±3.5.10-2 0.998 2.8 2.0 L-Gln*
D-Histidine 10.5 2.0–1000 91.3±3.0.10-5 -1.2±1.4.10-2 0.998 9.2 2.0 D-Ser*
D-Isoleucine 16.8 2.0–1000 84.7±4.6.10-4 8.4±2.1.10-1 0.995 0.075 2.0 D-Leu*
D-Leucine 17.2 2.0–1000 52.7±1.8.10-4 -6.6±8.3.10-2 0.998 4.0 2.0 D-Leu*
D-Lysine 20.5 2.0–1000 56.9±1.4.10-4 -7.3±6.4.10-2 0.9990 5.9 2.0 L-Lys*
D-Methionine 15.5 2.0–1000 45.6±1.1.10-4 -4.0±5.1.10-2 0.9990 10.0 2.0 D-Met*
D-Ornithine 20.4 2.0–1000 54.4±2.3.10-4 -1.2±1.1.10-2 0.997 0.20 2.0 L-Orn*
D-Proline 12.7 2.0–1000 82.2±2.7.10-4 0.1±1.3.10-1 0.998 0.75 2.0 D-Pro*
D-Serine 9.5 2.0–1000 47.2±1.2.10-4 3.4±5.3.10-2 0.9990 5.6 2.0 D-Ser*
D-Threonine 11.9 2.0–1000 77.6±2.3.10-4 0.1±1.1.10-1 0.998 1.2 2.0 L Thr*
D-Tryptophan 18.6 2.0–1000 92.6±3.8.10-4 -1.3±1.7.10-1 0.997 2.1 2.0 L Trp*
D-Tyrosine 21.4 2.0–1000 376.2±9.6.10-5 -1.4±4.4.10-2 0.9990 4.8 2.0 L Tyr*
D-Valine 15.2 2.0–1000 351.9±9.5.10-4 -1.2±4.4.10-2 0.9990 4.9 2.0 D-Val*

The calibration parameters in the study of L-amino acids.

Linearity study

RT (min) Range
(ppm;
µg/mL) Slope Intercept r IS

L-Alanine 9.9 0.200-10.0 6.08±0.21 -0.7±1.2 0.997 L-Ala*

L-Arginine 12.0 0.200-10.0 6.78±0.44 1.77±0.96 0.996 L-Arg*
L-Asparagine 7.3 0.200-10.0 2.96±0.22 0.93±0.55 0.993 L Asn*
Table 3:

L-Glutamine 8.3 0.200-10.0 5.24±0.16 -0.52±0.94 0.998 L-Gln*

L-Histidine 8.7 0.200-10.0 1.196±0.090 -0.39±0.42 0.990 L-Ser*
L-Isoleucine 15.0 0.200-10.0 7.42±0.63 2.2±1.6 0.990 L-Leu*
L-Leucine 15.4 0.200-10.0 3.615±0.092 1.06±0.46 0.9991 L-Leu*
L-Lysine 20.3 0.200-10.0 2.74±0.48 -1.4±3.2.10-2 0.990 L-Lys*
L-Methionine 14.1 0.200-10.0 4.70±0.29 -0.3±1.3 0.993 L-Met*
L-Ornithine 20.1 0.200-10.0 21.7±1.2 -4.1±5.6 0.995 L-Orn*
L-Proline 11.8 0.200-10.0 56.71±0.11 1.14±0.50 0.9994 L-Pro*
L-Serine 8.2 0.200-10.0 55.68±0.17 0.60±0.78 0.998 L-Ser*
L-Threonine 9.5 0.200-10.0 92.03±0.30 -0.70±1.4 0.998 L Thr*
L-Tryptophan 16.9 0.200-10.0 67.11±0.15 0.17±0.72 0.9991 L Trp*
L-Tyrosine 21.1 0.200-10.0 22.49±0.16 0.64±0.23 0.9990 L Tyr*
L-Valine 13.3 0.200-10.0 45.66±0.11 0.17±0.54 0.9990 L-Val*

Precision, accuracy and matrix effect in food samples.

Intra-day (n=3) Inter-day (n=6)

Concentratio Precisio Precisio Recover Instrumenta

n with n with
n Accurac Accurac y l Precision
samples samples
y (%) y (%)
Matrix RSD (%)
(ng/mL) RSD (%) RSD (%)
Effect (n=10)
Table 4:

D-Alanine 2.0 1.6 102.0 6.2 99.1 4.7% 0.83

20 3.1 99.2 3.8 99.0
100 2.0 103.6 2.2 104.2
250 2.9 103.3 3.6 105.8
D-Arginine 2.0 15.4 102.1 16.7 96.1 8.7% 1.6
10.0 4.1 113.0 10.3 109.6
50 5.7 106.4 4.8 109.0
125 1.1 108.0 3.0 110.7
D-Asparagine 2.0 2.9 100.8 7.2 96.7 4.5% 1.4
10 3.0 103.9 6.9 108.9
50 6.7 102.2 4.9 104.5
125 2.5 97.6 3.8 100.4
D-Cysteine (A) 40 2.2 96.5 8.4 104.0 6.6% 1.4

100 2.6 94.1 2.7 94.5

250 1.7 97.9 2.7 96.8
D-Glutamine 2.0 17.0 93.4 14.0 89.4 4.0% 1.2
10 6.4 98.1 9.8 97.0
70 5.3 96.0 6.2 100.6
200 2.2 94.1 2.9 92.5
D-Histidine 2.0 11.8 105.9 8.8 109.8 12.3% 2.4
20 7.0 102.9 8.5 96.9
100 6.9 105.5 11.0 97.9
250 4.4 104.6 3.1 104.3
D-Isoleucine 2.0 14.1 97.2 19.2 87.1 5.4% 0.73
20 2.4 97.6 5.5 93.2
100 8.7 84.0 9.8 78.5
250 8.4 87.8 15.6 79.4
D-Leucine 2.0 5.5 94.2 11.8 94.7 3.8% 0.67
20 1.3 94.4 4.9 92.6
100 0.8 100.6 3.1 102.5
250 3.1 103.2 2.5 102.2
D-Lysine 20 8.6 113.6 5.7 115.0 14.3 1.0
40 5.5 93.2 4.2 93.9
100 4.2 102.2 7.9 109.0
250 5.0 91.5 5.3 90.5
D-Methionine 2.0 1.4 98.2 5.9 97.8 2.7% 0.70
20 3.5 97.1 3.2 97.5
100 3.3 103.1 2.9 102.6
 250 3.1 97.5 4.8 101.3

D-Ornithine 2.0 11.5 86.6 18.0 102.0 13.7% 2.3

20 5.1 90.3 6.8 85.7
100 5.1 101.8 6.4 105.6
250 1.6 98.9 10.2 106.6
D-Proline 2.0 8.1 102.4 11.2 98.2 2.6% 0.86
20 5.0 93.9 4.9 93.9
100 4.3 100.9 3.8 101.3
250 4.9 102.4 4.8 99.4
D-Serine 2.0 7.4 101.4 11.4 101.0 6.6% 0.88
20 4.0 100.4 5.7 96.2
100 4.8 96.6 4.0 95.2
250 1.7 96.6 2.6 95.2
D-Threonine 2.0 2.9 109.0 5.9 106.6 2.3% 1.6
10 7.6 98.1 9.1 96.1
50 1.1 103.5 2.6 101.8
125 1.6 98.4 4.8 98.3
D-Tryptophan 2.0 5.0 115.2 7.3 118.5 6.9% 0.82
20 6.8 103.7 7.5 99.1
100 4.5 104.9 7.9 98.8
250 4.7 103.8 6.0 98.7
D-Tyrosine 5.0 7.5 104.5 11.6 115.5 6.4% 1.2
20 3.3 99.9 4.9 101.8
60 1.9 103.3 3.9 106.4
140 9.2 106.0 10.2 93.7
D-Valine 2.0 1.8 95.9 6.4 91.1 0.0% 1.5
20 4.9 97.6 5.8 99.7
100 4.4 102.9 5.7 106.8
250 7.1 102.3 6.7 106.3
 Table 5: Stability study: (A) Freshly prepared 200 ng/mL solution from a 1,000 ppm stock solution of the
labelled standards after 12 months. (B) With Post-derivatised cooked food samples at different times on
the autosampler tray at 12ºC. *D-Lys was with standard solution (not detected in sample).

(A)
TIME 0 12 months %D-AA*
Commercial AA* %D-AA* %D-AA* difference
d4-DL-Alanine 42.6% 46.7% 4.1%
N2-L-Arginine 0.0% 0.0% 0.0%
N2-L-Asparagine 0.0% 0.1% 0.1%
d5-L-Glutamine 0.0% 0.0% 0.0%
d10-DL-Leucine 60.2% 59.2% -1.0%
d4-L-Lysine 0.0% 0.1% 0.1%
d4-DL-Methionine 59.2% 64.3% 5.1%
d6-L-Ornithine 0.0% 28.0% 28.0%
d5-DL-Phenylalanine 90.4% 80.7% -9.7%
d7-DL-Proline 39.4% 42.4% 3.0%
d3-DL-Serine 49.4% 54.1% 4.7%
13
[ C4, N]-L-Threonine 3.2% 3.5% 0.3%
d5-L-Tryptophan 0.0% 1.8% 1.8%
d2-L-Tyrosine 0.0% 2.4% 2.4%
(B) d8-DL-Valine 35.2% 47.8% 12.6%
Variation (%) vs time

Stability D-Ala L-Ala D-Arg L-Arg D-Asn L-Asn D-Gln L-

time (h) Gln
10 1.1 0.5 4.6 -3.3 0.5 3.4
24 4.0 -1.1 -3.0 0.2 3.1 -4.9
36 5.5 -0.5 -4.7 -1.2 -4.2 3.8
-0.3
48 7.1 -1.7 -0.8 1.5 1.2 17.8 -3.9
60 7.9 -1.6 -3.8 0.1 1.0 13.8 0.6
Stability D-His L-His D-Ile L-Ile D-Leu L-Leu D-Lys* L-
time (h) Lys
10 4.6 1.8 -1.0 2.1 1.1 4.2 -7.0 7.9
24 8.9 8.5 0.5 -4.2 2.0 0.7 1.3
36 6.3 4.2 1.8 5.1 2.0 7.1
48 7.2 8.3 -7.5 2.8 0.1 0.2

60 11.3 6.3 2.6 2.8 2.0 -5.9 4.0

Stability D-Met L-Met D-Orn L-Orn D-Pro L-Pro D-Ser L-

time (h) Ser
10 -5.2 -1.4 -3.4 0.5 2.2 2.5 3.8 -2.2
24 6.7 -3.5 -1.0 4.9 -4.9 2.8 1.1 2.2
36 -2.7 -7.2 -6.0 0.2 2.0 4.0 6.3 2.2
48 -3.4 -4.1 13.9 11.6 -7.7 2.4 9.8 2.5
 60 -9.7 -7.4 8.1 9.0 -4.3 5.3 4.8 -1.1
Stability D-Thr L-Thr D-Trp L-Trp D-Tyr L-Tyr D-Val L-
time (h) Val
10 -8.2 -2.1 7.2 0.6 0.8 -1.7 5.2 -0.2
24 -4.0 0.0 -6.7 6.0 6.3 -0.1 5.4 0.3
36 -4.5 -2.2 5.8 3.3 10.6 -0.7 9.0 1.7
48 -6.6 0.7 9.8 5.7 1.4 -0.2
60 -1.8 -3.3 -1.4 9.1 -4.0 0.4 1.6 1.6

Table 6. Comparison of our method with other LC-MS/MS methods.

OUR METHOD
Derivatizatio ESI Elution Analysis Linearity Intra-day Inter-day
CDA n reaction Analytes Matrix Ionization IS order time Rs Sensitivity range (D-AA) precision precision
time mode
17 pairs of
2-1000 µg/L;
AAs 0.93 0.075-27.8
(S)-NIFE 20 min Cooked meals positive 15 AA* L<D 33 min Ɨ 40-1000 µg/L < 9.2 %Ɨ Ɨ < 11.8 % Ɨ Ɨ Ɨ
enantiomer - 6.0 pg (LOQ)
D-Cys
s
INDIRECT ANALYSIS

Rs

DerivatizatioESI
Elution Analysis Linearity Intra-day Inter-day
CDA n reaction Analytes Matrix Ionization IS Sensitivity order time range (D-AA) precision precision time mode
(R)-1-Boc-2-
 D-Arg piperidine 3.8 pmol 0.19 - 25
3h D-Ser human plasma positive (non- L<D 25 min 1.5 8.38% 8.38% carbonyl
(LOQ) µmol/L D-Ser labelled) chloride [24]

0.01 - 25
19 pairs of black vinegarb
µmol/L D-Trp;
(R)-BiACa [28] 10 min AAs and a lactic acid positive several D D<L 14.5 min > 3.6 - 127 0.025 - 50 < 12.4 % # -
enantiomer bacteria and L AA* 1.91 amol (LOD)
µmol/L D-
Ala s beverage and D-Tyr
2 - 2000
1.62 nmol/L; urine/plasma/CS 0.34 -
51.5 0.7 - 14.0
(S)-NIFE [46] 10 min 19 D-AAs positive 19 AA* L<D 21 min - 20-20,000 nM 2.1- 18 %
F pmol (LOD) %
11.17 D-Ala and D-
Ser
0.03 - 10 µg/L
18 AAs
0.15 pg D-Met; 20-
(S)-NIFE [42] 10 min enantiomer milk positive 19 AA* L<D 24 min - < 6.4 % -
(LOQ) 1000 µg/L Ds
Ser and D-Cys
0.063 – 25.1
# µg/L D-Leu
parts of rat brain 0.12- 20 pg
(S)-NIFE [43] 20 min 11 D-AAs # positive fudosteine L<D 18 min - and D-Ile; 5.0 < 10 % < 11 %
(LOQ)
– 2017 µg/L D-
Val
0.051 –5.1
0.1- 80 pg
(S)-NIFE [44] 20 min 18 D-AAs rat plasma positive fudosteine L<D 18 min - µg/L D-Asp; < 12.3 % < 10.1 %
(LOQ)
41.6 – 4164
µg/L D-Val

44.4-3294
amol
1.2 - (method A)
9 (A) and
19 pairs of 38 min and 92.15556
DMT-(S)-Pro- AAs (A) and 0.5 - amol
40 min enantiomer L-Ala-d3 or 26 min 10 (method B) 0.5 – 10
Osu [45] s Human saliva positive D-Ala-d3 L<D (B) (B) (LOD) µmol/L D-Ala < 7.4 % < 11.5 %
Marfey’s Ser 3-13C-DLSer
enantiomer cerebrospinal and 0.2 µmol/L 0.2 - 22
30 min negative L<D 15 min - <2% <6%
s and Gly fluid 1,2-13C2Gly (LLOQ) µmol/L D-Ser
reagent [47]
19 pairs of 0.82 1.7-7.9
Marfey's pmol
AAs 13 - LLOQ - 40
24 h - negative C3-D-Ala L<D 23 min (LLOQ) - -
enantiomer 4.42 µmol/L L-AAs
reagent [48]
s and Gly
DIRECT ANALYSIS

Derivatizatio ESI Elution Analysis Linearity Intra-day Inter-day

CDA n reaction Analytes Matrix Ionization IS order time Rs Sensitivity range (D-AA) precision precision
time mode
19 AAs
enantiomer
s and Gly 0.625-25 nM
DIRECT L-Metd3,L-
2.25- 0.6-22.8 D-Trp 500-
NA (except for human serum positive Phe- D<L 10.5 <14.4% <14.1%
25.96 amol (LOD) 25,000 nM D-
ANALYSIS [32] DL-Pro, L- d2
Ile
Gln, and D-
Lys)
2D - LC MS/MS

0.005 pmol 0.0005 - 5 µM

4-fluoro-7nitro-2,1,3- 6 D-AAs
2 min D-Ala, D-Ala, D-Leu,
(Ala,Asp, ¥ 1.87– 0.58–7.18 1.37–8.37
Urine and plasma positive none D<L 515 min DLeu, D-Pro D-Pro and D-
benzoxadiazol e (NBD-F) Glu, Leu, 5.17 % %
and D-Ser; Ser; 0.005 - 1
[49] Pro and Ser)
0.05 pmol
 D-Asp; 0.01 µM D-Asp;
pmol D-Glu 0.001 - 5 µM
(LOQ) D-Glu

a b

: (R)-4-nitrophenyl N-*2′-(diethylamino)-6,6′-dimethyl-*1,1′-biphenyl]-2-yl]carbamate hydrochloride; : Japanese traditional Kurozu; (A): EBH

C18 column; (B): ADME column; AA*: labelled AAs; Rs: resolution between D and L isomers; IS: internal standard. Ɨ Rs = 11.0 for d5-DL-Phe Ɨ Ɨ
At 2.0 ng/mL: D-Arg= 15.4 %, D-Gln=17.0 %, D-His= 11.8 %, D-Ile= 14.1 %, D=Orn 11.5 % ; Ɨ Ɨ Ɨ At 2.0 ng/mL: D-Arg= 16.7 %, D-Gln=14.0 %, D-
Ile= 19.2 %, D=Orn 18.0 % and at 250 ng/mL D-Ile= 15.6 %; # in the low quality control 20.2% for L-Ser and 22.8% for Gly; # # cortex,
hippocampus, cerebellum and olfactory bulb; ¥ KSAARP column (1.0 mm i.d. x 500 mm an original ODS column designed by collaboration with
Shiseido) and a KSAACSP001S column (1.5 mm i.d. x 250 mm, an original Pirkle-type column produced by collaboration with Shiseido).
Table 7: Concentrations of AAs (µmol/g) and % of D in the pool of food samples used for the validation (n=6). SEM:
Standard error of the mean. ND: not detected.
D-AA Average ± SEM L-AA Average ± SEM D/(D+L)-AA Average (%) ± SEM
D-Ala 8.02±0.23 L-Ala 1648.3±36.2 D/(D+L)-Ala 0.4838±0.0056
D-Arg 169.4±3.6·10 -3
L-Arg 183.6±1.3 D/(D+L)-Arg 0.0922±0.0020
D-Asn 2.65±0.091 L-Asn 645.8±3.3 D/(D+L)-Asn 0.405±0.012
D-Cys 4.45±0.41 L-Cys 40.2±2.1 D/(D+L)-Cys 10.05±0.97
D-Gln 0.803±0.032 L-Gln 393.2±8.6 D/(D+L)-Gln 0.2034±0.0044
D-His 57.2±3.1 L-His 155.0±1.6 D/(D+L)-His 26.7±1.1
D-Ile 0.644±0.056 L-Ile 132.25±0.50 D/(D+L)-Ile 0.485±0.044
D-Leu 0.753±0.017 L-Leu 170.5±2.5 D/(D+L)-Leu 0.4392±0.0061
D-Lys ND L-Lys 1112.1±25.3 D/(D+L)-Lys ND
D-Met 44.6±1.8·10-3 L-Met 245.2±4.2 D/(D+L)-Met 0.01814±0.00051
D-Orn 0.189±0.059 L-Orn 61.7±1.4 D/(D+L)-Orn 0.308±0.095
D-Pro 1.300±0.052 L-Pro 698.5±7.3 D/(D+L)-Pro 0.1857±0.0071
D-Ser 5.72±0.17 L-Ser 618.1±12.0 D/(D+L)-Ser 0.917±0.015
D-Thr 0.322±0.013 L-Thr 429.6±8.9 D/(D+L)-Thr 0.0751±0.0030
D-Trp 35.7±1.5·10-3 L-Trp 97.8±1.9 D/(D+L)-Trp 0.0364±0.0011
D-Tyr ND L-Tyr 110.9±1.0 D/(D+L)-Tyr ND
D-Val 334.8±6.4·10-3 L-Val 551.1±8.2 D/(D+L)-Val 0.06072±0.00097

43