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MS quantification of carnosine, anserine, and balenine in meat samples
Journal Pre-proofs LC-ESI-MS/MS quantification of carnosine, anserine, and balenine in meat samples Reiko Uenoyama, Masao Miyazaki, Tamako Miyazaki, Yuhei Shigeno, Yoshinori Tokairin, Hiroyuki Konno, Tetsuro Yamashita PII: DOI: Reference:
S1570-0232(19)30830-X https://doi.org/10.1016/j.jchromb.2019.121826 CHROMB 121826
To appear in:
Journal of Chromatography B
Received Date: Revised Date: Accepted Date:
24 May 2019 6 October 2019 8 October 2019
Please cite this article as: R. Uenoyama, M. Miyazaki, T. Miyazaki, Y. Shigeno, Y. Tokairin, H. Konno, T. Yamashita, LC-ESI-MS/MS quantification of carnosine, anserine, and balenine in meat samples, Journal of Chromatography B (2019), doi: https://doi.org/10.1016/j.jchromb.2019.121826
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© 2019 Published by Elsevier B.V.
LC-ESI-MS/MS quantification of carnosine, anserine, and balenine in meat samples Reiko Uenoyama1, Masao Miyazaki1, Tamako Miyazaki1, Yuhei Shigeno2, Yoshinori Tokairin2, Hiroyuki Konno2, and Tetsuro Yamashita1
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Department of Biological Chemistry and Food Sciences, Faculty of Agriculture, Iwate
University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan 2
Department of Biochemical Engineering, Graduate School of Science and Engineering,
Yamagata University, 4-3-16 Jounan, Yonezawa, Yamagata 992-0038, Japan
*Corresponding author. [email protected] (Tetsuro Yamashita)
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Abstract The histidine-containing imidazole dipeptide carnosine and its methylated analogs anserine and balenine are present at high concentrations in vertebrate tissues. Although the physiological functions of the imidazole dipeptides have not been elucidated yet, it has been suggested that they play significant biological roles in animals. Despite increasing interest, few studies have challenged the quantifications of carnosine, anserine, and balenine by a single HPLC run because they have similar retention times. In this study, we developed a method to quantify these imidazole dipeptides in meat samples using an LC-ESI-MS/MS triple-quadrupole mass spectrometer. We improved the liquid chromatographic separation of the imidazole peptides by applying a mix-mode column, which provides both normal phase and ion exchange separations, and developed multiple reaction-monitoring of the transitions for quantification of m/z 227 → 110 for carnosine, m/z 241 → 126 for anserine, m/z 241 → 124 for balenine, and m/z 269 → 110 for L-histidyl-L-leucine (internal standard). The established method met all pre-defined validation criteria. Intra- and inter-day accuracy and precision were ± 10.0% and ≤ 14.8%, respectively. The ranges of quantifications were 14.7 ng/mL–1.5 mg/mL for carnosine, 15.6 ng/mL–1.6 mg/mL for anserine, and 15.6 ng/mL–1.6 mg/mL for balenine. In conclusion, the validated method was successfully applied to the quantification of imidazole dipeptides in biological samples without derivatization.
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1. Introduction Carnosine (β-alanylhistidine, Fig. 1A) and its methylated analogs anserine (β-alanyl-1methylhistidine, Fig. 1B) and balenine (β-alanyl-3-methylhistidine, ophidine, Fig. 1C) are histidine-containing imidazole dipeptides, which are distributed in vertebrate tissues, especially in the skeletal muscle [1], the heart, and the brain [2, 3]. Although the physiological functions of these dipeptides have not been clearly elucidated [4], it has been suggested that the imidazole dipeptides function as buffers against the accumulation of lactic acid in muscle tissues [5], as inducers of enzymes expressed in muscles [6], as precursors for the synthesis of neurotransmitters [7], and as scavengers of free radicals [8]. The ratio of the imidazole dipeptides is characteristic in meat samples of different species. Anserine and balenine are present at higher concentrations (up to 10 mM) compared with other imidazole dipeptides in chicken and whale meat samples, respectively [3, 4, 9]. Unlike meat proteins that are degraded during cooking, the imidazole dipeptides are not degraded because they do not have a secondary or tertiary structure; thus, they are suitable targets for species identification [5, 9]. Quantifying the tissue contents of the imidazole dipeptides will be useful in identifying the species from which processed meats originated as an anti-counterfeiting method [5, 9]. High-performance liquid chromatography (HPLC) allows the analytical separation of the imidazole dipeptides contained in tissue samples. Reverse-phase (RP)-HPLC and hydrophilic interaction chromatography (HILIC) have been applied to separate the imidazole dipeptides in tissue samples, followed by detection using ultraviolet (UV) and fluorescence detectors [3, 10-12]. RP-HPLC is a powerful separation technique for various low molecular weight organic compounds, and results in better separation of the compounds compared with HILIC [13]. However, one disadvantage of RP-HPLC is the low retention time of polar molecules, including the imidazole dipeptides [14]. Therefore, it is necessary to derivatize the
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dipeptides before RP-HPLC [15], although this derivatization process is time-consuming. Moreover, detection using UV spectroscopy of the imidazole dipeptides has several disadvantages, including lower selectivity and a higher value of lower limit of quantification (LLOQ) compared to the fluorescence detector. However, for fluorescence detection of the dipeptides, fluorescence labeling of the samples is necessary for HPLC sample preparations. In the last dozen years, electrospray ionization-mass spectrometry (ESI-MS) has been used as a detection method for the analysis of the imidazole dipeptides [16, 17]. Anserine and balenine, which are structural isomers, were quantified despite the subtle difference of these structures using an LC-MS system consisting of RP-HPLC and an ESI-ion trap mass spectrometer (ITMS) [18]. However, the LC-ESI-ITMS system was necessary to carry out MS/MS/MS (MS3) of anserine and balenine for discriminating them; MS/MS (MS2) of them could not adequately discriminate them. To overcome these issues, this study aimed to optimize liquid chromatography-ESItandem mass spectrometry (LC-ESI-MS/MS) for the quantification of carnosine, anserine, and balenine simultaneously in tissue samples. To improve separation of the imidazole dipeptides, we applied a mix-mode column, which provides both normal phase and ion exchange separations to HPLC. Furthermore, we used a triple-quadrupole mass spectrometer for quantifications of the imidazole dipeptides. This method shows higher selectivities and better LLOQs for the quantification of carnosine, anserine, and balenine without derivatization compared to previous methods described above.
2. Materials and Methods 2.1. Chemicals Acetonitrile (LC-MS grade, ≥ 99.9% purity) was purchased from Honeywell (Morristown, NJ, USA). Formic acid (LC/MS grade, 99% purity), 1 M ammonium formate
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solution (HPLC grade), ethanol (reagent grade, 99.5% purity), chloroform (reagent grade, 99.0% purity), L-carnosine (≥ 98.0% purity), and L-anserine nitrate (≥ 95.0% purity) were purchased from Wako Pure Chemical Corp. (Osaka, Japan). L-Balenine (99.9% purity) was purchased from Hamari Chemicals, Ltd. (Osaka, Japan). L-Histidyl-L-leucine (His-Leu, ≥ 99.0% purity, Fig. 1D) was purchased from Peptide Institute, Inc. (Osaka, Japan).
2.2. Preparation of stock solution, calibration standards, and quality control samples Stock solutions of carnosine, anserine, and balenine were prepared in Milli-Q (MQ) water at concentrations of 2.26, 2.40, and 2.40 mg/mL, respectively. The stock solution of His-Leu (internal standard [IS]) was prepared in MQ water at a concentration of 0.67 mg/mL. Working solutions were prepared by mixing the stock solutions and diluting with 0.1 M HCl in MQ water. The stock and working solutions were stored at –20°C until analysis. The carnosine, anserine, and balenine concentrations in the calibration standard solutions were (14.7, 15.6, and 15.6 ng/mL), (58.8, 62.4, and 62.4 ng/mL), (236, 250, and 250 ng/mL), (942, 1,000, and 1,000 ng/mL), (3,770, 4,000, and 4,000 ng/mL), and (15,100, 16,000, and 16,000 ng/mL), respectively. The carnosine, anserine, and balenine concentrations were (14.7, 15.6, and 15.6 ng/mL) for the LLOQ quality control (QC) samples, (29.4, 31.2, and 31.2 ng/mL) for the low QC samples, (942, 1,000, and 1,000 ng/mL) for the medium (mid) QC samples, and (11,300, 12,000, and 12,000 ng/mL) for the high QC samples. These calibration standard solutions and QC samples were spiked with the IS at 17.9 ng/mL or 1,790 ng/mL as described below.
2.3. Meat samples Six chicken and six beef samples were obtained from Japanese supermarkets. We also used a meat sample from Sei whale (Balaenoptera borealis) caught by whaling for scientific
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purposes during the summer season in the Northwestern Pacific in 2017 under National Oceanic and Atmospheric Administration/National Marine Fisheries Service permission.
2.4. Sample preparation An aliquot (5.0 g) of each lean meat was minced with scissors and homogenized on ice with 20 mL of ethanol spiked with 134 μg of IS. The homogenate was centrifuged at 3,000 rpm for 10 min at 4°C. The supernatant was transferred to a separating funnel. The precipitate was rehomogenized with 20 mL of 80% ethanol in MQ water and, after centrifugation, the supernatant was transferred to the separating funnel; this process was repeated twice to improve the recovery rate. Then, 60 mL of chloroform was added to the supernatant, which was shaken until only a single layer was present. After adding 40 mL of MQ water, the separating funnel was shaken for 2 min to separate the aqueous and chloroform layers. The upper aqueous layer was transferred to a recovery flask, and then, after removing ethanol by rotary evaporation, was diluted to 75 mL with HCl in MQ water (the final concentration of HCl was 0.1 M). The solution was filtered through a 0.45-μm filter (Merck Millipore, Burlington, MA, USA) and transferred into a clean autosampler vial with low peptide adsorption (PSVial100; AMR, Inc., Tokyo, Japan). The meat samples that were diluted 100fold with 0.1 M HCl in MQ water were also analyzed. Each meat sample was analyzed in three independent LC-ESI-MS/MS experiments.
2.5. LC-ESI-MS/MS This study used the LCMS-8040 system (Shimadzu Co., Kyoto, Japan). For chromatographic separation of the imidazole dipeptides, the mix-mode column (Intrada Amino Acid; 3 μm; 3 mm i.d. × 50 mm; Imtakt, Kyoto, Japan) was used at 40°C. Each sample (15 μL) was injected with an autosampler (SIC-30AC; Shimadzu Co.) that was set at
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15°C. The needle of the autosampler was washed with MQ water. Separations were performed using the mobile phases of solvent A (0.1% formic acid in acetonitrile) and solvent B (20% acetonitrile and 80 mM ammonium acetate in MQ water) at a flow rate of 0.4 mL/min. The time program was as follows: 50% B for 14 min, 100% B for 2 min, and 50% for 5 min, for a total run time of 21 min. The effluent from the HPLC step was introduced online into a triple-quadrupole mass spectrometer. The mass spectrometer was operated in the multiple reaction-monitoring (MRM) mode using the following parameters: 100 ms of dwell time, 230 kPa of collision-induced dissociation gas, ESI in positive mode, 3 L/min of nebulizer gas, and 15 L/min of drying gas heated at 250°C. Data acquisition and processing were performed by LabSolutions (version 5.97, Shimadzu Co.).
2.6. Method validation The analytical method validation was conducted based on the bioanalytical method validation guidelines of the U.S. Food and Drug Administration [19]. The following validation parameters were verified: accuracy and precision, selectivity, matrix effect, recovery, stability, carryover, calibration model, and LLOQ. Values of accuracy and precision, calibration model, and LLOQ were calculated by LabSolutions (version 5.97, Shimadzu Co.).
2.6.1. Accuracy and precision Accuracy and precision were evaluated by analyzing five replicates of LLOQ, low, mid, and high QC samples in three independent validation runs. Intra- and inter-day accuracy values were described as the relative error [RE (%)]. The intra- and inter-day precision values were expressed as the coefficient of variation [CV (%)]. At each concentration level, the accuracy and precision were required to be within ± 15% and ≤ 15%, respectively. For the
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LLOQ, the accuracy and precision were required to be within ± 20% and ≤ 20%, respectively.
2.6.2. Selectivity and matrix effect To investigate the selectivity of the imidazole dipeptides that are endogenous compounds in meat samples, the potential interferences of endogenous substances were evaluated in six chicken, six beef, and one whale meat samples by calculating the relative difference of the ion ratio (%) as follows [20]: The relative difference of the ion ratio =
Ion ratio in meat samples – Ion ratio in QC samples × 100 (%) Ion ratio in QC samples
, where the ion ratio was calculated as follows: Ion ratio =
Peak area of the ion for quantification Peak area of the ion for qualification
The cross-analyte/IS interference was assessed by comparing the retention time between each imidazole dipeptide and IS. The matrix effect was verified in six chicken and six beef samples. To assess matrix effects, authentic carnosine (2,260 ng), anserine (2,400 ng), and balenine (2,400 ng) were spiked in 1 mL of 100-fold-diluted meat samples. Since the imidazole dipeptides are present at high concentrations in meat samples, matrix effects were calculated as follows: Matrix effect Peak areas of each measured analyte concentration in meat samples spiked with authentic analytes – Peak areas of each measured endogenous analyte concentration in meat samples = Peak areas of each measured spiked value of the authentic analytes in 0.1 M HCl in MQ water The matrix effect was considered acceptable if the precision [CV (%)] in the six chicken and six beef samples was ≤ 15%.
2.6.3. Recovery and stability
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The extraction recovery was evaluated by the recovery of the IS; i.e., the peak area ratio of the IS in six chicken and six beef samples prepared with 100-fold dilution which were spiked IS before extraction to in these which were spiked IS after extraction (IS concentration, 17.9 ng/mL) in triplicate. The stabilities of carnosine, anserine, and balenine were assessed after freezing and thawing fresh QC samples three times and after incubating fresh QC samples at 15°C for 72 h in the autosampler. These stability analyses were performed using low and high QC samples in triplicate. Each analyte was considered stable if its measured concentration after the treatments was within ± 15% of its measured concentration in the fresh QC samples.
2.6.4. Carryover For the assessment of carryover effects, the blank sample (0.1 M HCl in MQ water) was twice analyzed by the LC-ESI-MS/MS system immediately following analysis of an upper LOQ (ULOQ) sample spiked with the IS (final concentration, 1,790 ng/mL) in each analytical run.
2.6.5. Calibration Calibration curves were generated by calculating the peak area ratios of carnosine, anserine, and balenine to the IS versus the concentrations with a 1/concentration2 weighting factor. At least 75% of the non-zero standards (including the LLOQ and ULOQ standards) in every run were required to be ± 15% of their nominal value (± 20% for LLOQ samples). The correlation coefficient was calculated for each run.
2.6.6. LLOQ The signal-to-noise (S/N) ratio was calculated by LabSolutions software using the
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ASTM algorithm. The LLOQ of the assay was assessed in each analytical run at an S/N ratio greater than 10.
2.7. Statistical analysis Statistical significance was tested using the non-parametric Wilcoxon matched-pair signed rank test by JMP software (ver. 12.0; SAS Institute, Cary, NC, USA).
3. Results and Discussion 3.1. MRM transitions of carnosine, anserine, and balenine Product ion scans of carnosine, anserine, and balenine were carried out to establish the MRM transitions of these analytes. Each compound was introduced directly into the ESIMS/MS using the solvent (36% acetonitrile, 0.02% formic acid, and 64 mM ammonium acetate in MQ water), and fragmentation patterns were compared among these analytes. In the MS/MS spectrum of a precursor ion of carnosine, [M+H]+ at m/z 227, major product ions were detected at m/z 210, 181, 164, 156, and 110 (Fig. 2A), as described previously [21]. The previous study reported that an ion detected at m/z 110 (Fig. 2E, a) is produced by the loss of H2O and CO from an ion with m/z 156 (Fig. 2E, b), which corresponds to a protonated histidine. Since MS/MS analysis of carnosine exhibited its highest ion at m/z 110, this ion was selected for the quantification of carnosine using the MRM method. The ion at m/z 156 was selected for the qualification of carnosine. In the MS/MS spectrum of a precursor ion of anserine, a 1-methylated analog of carnosine with [M+H]+ at m/z 241, major product ions were detected at m/z 170, 153, 126, 124, 109, 96, and 68 (Fig. 2B). On the other hand, in the MS/MS spectrum of a precursor ion of balenine, a 3-methylated analog of carnosine with [M+H]+ at m/z 241, major product ions were detected at m/z 224, 195, 178, 170, 136, 124, and 109 (Fig. 2C). Notably, the MS/MS
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fragmentations of the product ions of anserine and balenine were different. In agreement with Turecek et al. [22], an ion detected at m/z 126 in the MS/MS spectrum of anserine (Fig. 2E, c) was produced by the loss of CO2 from an ion with m/z 170 (Fig. 2E, d), which corresponds to protonated 1-methylhistidine. By contrast, an ion detected at m/z 124 in the MS/MS spectrum of balenine (Fig. 2E, e) was produced by the loss of H2O and CO from an ion with m/z 170 (Fig. 2E, f), which corresponds to protonated 3-methylhistidine. These results suggest that the difference in methylated positions between anserine and balenine caused the difference in their MS/MS fragmentations. More specifically, in balenine, an amide proton may be easily activated by a nitrogen at position 1 of the imidazole ring since the amide proton and the imidazole nitrogen form a six-membered ring; thus, an imine would be formed by the desorption of CO2. By contrast, the amide proton of anserine would not likely be activated by the imidazole nitrogen because of steric hindrance caused by binding of the methyl group to the imidazole nitrogen; thus, an amine state is maintained, even with the loss of CO2. Since MS/MS analysis of balenine produced the highest ion at m/z 124 (Fig. 2E, e), we selected this ion for the quantification of balenine. The ion at m/z 224 was detected only from balenine, and not from anserine, hence this ion was selected for qualification of balenine. Although MS/MS analysis of anserine produced the highest ion at m/z 109, this ion was also detected as a major ion in balenine. Therefore, the other ion detected at m/z 126 was selected for the quantification of anserine. Although the ion at m/z 153 was detected only for anserine but not from balenine, its intensity was lower than that of the ion at m/z 126. Therefore, the ion detected at m/z 153 was selected for the qualification of anserine. The MRM transitions of carnosine, anserine, and balenine for quantifications and qualifications are shown in Table 1. The collision energies were optimized to generate the highest signal intensities of the ions to be quantified and qualified.
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3.2. LC-ESI-MS/MS analyses of carnosine, anserine, and balenine The MS/MS chromatograms of the imidazole dipeptides are illustrated in Fig. 3. In the LC-ESI-MS/MS analysis of the mid QC samples, balenine (C), anserine (B), and carnosine (A) were eluted at 9.4, 10.9, and 11.6 min, respectively. The ions for the quantification of balenine (m/z 241 → 124) and anserine (m/z 241 → 126) were also detected as minor peaks at 10.9 and 9.4 min, respectively. This is because the precursor ions of balenine and anserine produced MS/MS fragment ions at m/z 126 and m/z 124, respectively. However, since balenine and anserine were not co-eluted in the HPLC system with the mix-mode column, we concluded that ions with m/z 241 → 124 and m/z 241 → 126 are useful to quantify balenine and anserine, respectively.
3.3. Internal standard selection In our work, His-Leu was selected as the IS because it is an imidazole dipeptide. In the MS/MS spectrum of the His-Leu precursor ion of [M+H]+ at m/z 269, a major product ion was detected at m/z 110 (Fig. 2D). His-Leu displayed similar chromatographic behavior as the other imidazole dipeptides, but the retention time (7.8 min) was different, indicating that there was no cross-analyte/IS interference with this method. Furthermore, the MS/MS chromatogram of m/z 269 → 110 for the IS was compared between the IS-free meat samples and the calibration/QC samples containing 17.9 ng/mL of IS (Fig. 4). No interferences of endogenous substances to the IS were confirmed based on the result that the IS responses in the IS-free meat samples were ≤ 5% of the average IS responses of the calibration and QC samples. Thus, the m/z 269 → 110 ion was selected for the quantification of His-Leu.
3.4. Method validation 3.4.1. Accuracy and precision
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The accuracy and precision results for quantification of the imidazole dipeptides are shown in Table 2. The intra- and inter-day accuracy values of the imidazole dipeptides were within ± 5.4% and ± 9.0%, respectively, for all of the low, mid, and high QC samples. The intra- and inter-day precision values were ≤ 4.9% and ≤ 14.8%, respectively. For the LLOQ samples, the intra- and inter-day accuracy values were within ± 2.7% and ± 2.0%, respectively. The intra- and inter-day precision values were ≤ 10.0% and ≤ 17.1%, respectively. These results indicated that the established method for quantification of the imidazole dipeptides is highly accurate and precise.
3.4.2. Selectivity and matrix effect Since the imidazole dipeptides are contained as endogenous compounds in meat samples, it was difficult to evaluate their selectivity using blank matrix samples. Therefore, we evaluated the relative difference of the ion ratio of each imidazole dipeptide to establish the selectivity (Table 3). The relative difference of the imidazole dipeptides was within ± 0.26% for all of the meat samples. These results indicated that no significant interference with endogenous substances contained in meat samples was found at the retention time of each analyte. The matrix effects of six chicken and six beef samples on the imidazole dipeptide are shown in Table 4. All of the matrix effects were ≤ 6.0%, indicating that there was no matrix effect of the developed method for quantification of the imidazole dipeptides.
3.4.3. Recovery and stability The mean extraction recovery values of the IS were 87.5 ± 2.5% and 85.8 ± 9.9% in six chicken and six beef samples, respectively. The stabilities of the imidazole dipeptides are shown in Table 5. The imidazole dipeptides of the low and high QC samples were stable after
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three freeze/thaw cycles and incubation at 15°C for 72 h.
3.4.5. Carryover No carryovers of the imidazole dipeptides and the IS were observed in the first blank samples after analyzing an ULOQ sample containing 1,790 ng/mL of the IS. Therefore, the concentrations of the imidazole dipeptides under the ULOQ were appropriate to quantify without a carryover effect.
3.4.6. Calibration and LLOQ Our preliminary experiments showed that the balenine content was approximately 80fold higher than that of anserine in the whale meat samples, and the carnosine content was approximately 160-fold higher than that of balenine in the beef samples. Therefore, to quantify the contents of each imidazole dipeptide in meat samples, we analyzed both meat samples with and without 100-fold dilution. Since the final concentrations of the IS were different between meat samples with and without the dilution, we generated two calibration curves based on the different IS concentrations. Carnosine, anserine, and balenine with low concentrations (< 1,470, < 1,560, and < 1,560 ng/mL) in meat samples were quantified without dilution using a calibration curve based on 1,790 ng/mL of the His-Leu IS. Carnosine, anserine, and balenine with high concentrations (> 1,470, > 1,560, and > 1,560 ng/mL) in meat samples were quantified with 100-fold dilution using a calibration curve based on 17.9 ng/mL of the His-Leu IS. Table 6 shows that the ranges of concentration for the two calibration curves were 14.7 ng/mL–15.1 μg/mL for carnosine and 15.6 ng/mL–16.0 μg/mL for anserine and balenine. Linearity was acceptable with the correlation coefficient (r2) exceeding 0.994 for the imidazole dipeptides.
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In previous studies using LC-ESI-ITMS to analyze the imidazole dipeptides in meat samples, LLOQ was 12.5 pmol for each imidazole dipeptide [18]. In this study using LCESI-MS/MS to quantify the imidazole dipeptides, the LLOQs (S/N > 10:1) were 14.7 ng/mL for carnosine, 15.6 ng/mL for anserine, and 15.6 ng/mL for balenine, which convert to approximately 1 pmol injected for each imidazole dipeptide (Table 6). LC-ESI-MS/MS analysis of meat samples, using samples that were prepared at 100-fold dilution, enabled quantification of the imidazole dipeptides over the wide ranges of 14.7 ng/mL–1.5 mg/mL for carnosine, 15.6 ng/mL–1.6 mg/mL for anserine, and 15.6 ng/mL–1.6 mg/mL for balenine. These ranges convert to 0.977 nmol/g–100 μmol/g of wet mass tissue for the imidazole dipeptides, which was wider than the range of the previous reports, (15–1,000 nmol/g) [18] and (0.1–0.8 μg/mL for L-carnosine) [23]. These method validation results indicate that the developed method improves selectivity and the LLOQ of anserine, carnosine, and balenine, and provides wider concentration ranges compared with the previous researches [18, 23].
3.5. Quantification of carnosine, anserine, and balenine in meat samples Fig. 5 shows representative MS chromatograms of the imidazole dipeptides in the meat samples. For the quantifications of carnosine, anserine, and balenine, adequate separations of the imidazole dipeptides were achieved in a single run of each meat sample as well as the mid QC sample. A recent study applied a chirobiotic T column which contains the glycopeptide teicoplanin covalently bonded to a high purity spherical silica gel [23]. As compared to the chirobiotic T column, the mix-mode column providing both normal phase and ion exchange separations shows the better separation for the imidazole dipeptide. Thus, the validated method was applied to quantify the imidazole dipeptides in the chicken, beef, and whale meat samples. For quantification of the imidazole dipeptides in the meat samples, 100-fold dilution
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was necessary to obtain values within the calibration range, except for balenine in the chicken and beef samples. Fig. 6 shows the quantification results for the imidazole dipeptides in the meat samples for the three independent LC-ESI-MS/MS runs. Good repeatability was observed for each sample. The content of each imidazole dipeptide per gram of wet meat followed a non-normal distribution in the three types of meats. Thus, the contents were evaluated using the nonparametric Wilcoxon matched-pair signed rank test. Beef and chicken samples showed significant differences among contents of the imidazole dipeptides (p<0.0001). The highest contents in chicken and beef samples corresponded to anserine and carnosine, respectively. Balenine had the lowest content in the chicken and beef samples, in contrast to a whale meat sample. Each imidazole dipeptide content was different within a given type of meat. Table 7 shows the contents of the imidazole dipeptides in meat samples reported in other studies [3, 5, 10, 24, 25]. Our quantified contents are consistent with those data. Therefore, we propose that our developed method can be used to quantify the imidazole dipeptides in meat samples.
4. Conclusions We developed a quantification method for carnosine, anserine, and balenine in meat samples using both HPLC with a mix-mode column and ESI triple-quadrupole mass spectrometer. The validated method exhibits high selectivity, low LLOQ, and wide ranges of concentrations for the simultaneous quantification of the imidazole dipeptides in biological samples. This optimized method will contribute to the anti-counterfeiting method and the elucidation of biological functions of imidazole dipeptides in vertebrates.
Conflicts of interest
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The authors declare they have no actual or potential competing conflict of interest.
Acknowledgement We thank Ms. Manami Kobayashi of Shimadzu Co. for experimental supports.
References [1] H. Stuerenburg, The roles of carnosine in aging of skeletal muscle and in neuromuscular diseases, Biochemistry (Moscow), 65 (2000) 862-865. [2] L. Bonfanti, P. Peretto, S. De Marchis, A. Fasolo, Carnosine-related dipeptides in the mammalian brain, Progress in Neurobiology, 59 (1999) 333-353. [3] L. Mora, M.A. Sentandreu, F. Toldra, Hydrophilic chromatographic determination of carnosine, anserine, balenine, creatine, and creatinine, Journal of Agricultural and Food Chemistry, 55 (2007) 4664-4669. [4] T. Nagasawa, T. Yonekura, N. Nishizawa, D.D. Kitts, In vitro and in vivo inhibition of muscle lipid and protein oxidation by carnosine,
, 225
(2001) 29-34. [5] P.R. Carnegie, M.Z. Ilic, M.O. Etheridge, M.G. Collins, Improved high-performance liquid chromatographic method for analysis of histidine dipeptides anserine, carnosine and balenine present in fresh meat, Journal of Chromatography A, 261 (1983) 153-157. [6] P. Johnson, J.L. Hammer, Effects of L-1-methyl-histidine and the muscle dipeptides carnosine and anserine on the activities of muscle calpains, Comparative Biochemistry and Physiology. B, 94 (1989) 45-48. [7] E.A. Decker, A.D. Crum, J.T. Calvert, Differences in the antioxidant mechanism of carnosine in the presence of copper and iron, Journal of Agricultural and Food Chemistry, 40 (1992) 756-759.
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[8] E.A. Decker, The role of phenolics, conjugated linoleic acid, carnosine, and pyrroloquinoline quinone as nonessential dietary antioxidants, Nutrition Reviews, 53 (1995) 49-58. [9] P.R. Carnegie, K.P. Hee, A.W. Bell, Ophidine (β-alanyl-L-3-methylhistidine, ‘balenine’) and other histidine dipeptides in pig muscles and tinned hams, Journal of the Science of Food and Agriculture, 33 (1982) 795-801. [10] M.C. Aristoy, F. Toldrá, Histidine dipeptides HPLC-based test for the detection of mammalian origin proteins in feeds for ruminants, Meat Science, 67 (2004) 211-217. [11] M. Dunnett, R.C. Harris, High-performance liquid chromatographic determination of imidazole dipeptides, histidine, 1-methylhistidine and 3-methylhistidine in equine and camel muscle and individual muscle fibres, Journal of Chromatography B, 688 (1997) 47-55. [12] L. Mora, A.S. Hernández-Cázares, M.-C. Aristoy, F. Toldrá, M. Reig, Hydrophilic interaction chromatography (HILIC) in the analysis of relevant quality and safety biochemical compounds in meat, poultry and processed meats, Food Analytical Methods, 4 (2011) 121-129. [13] T. Ikegami, K. Tomomatsu, H. Takubo, K. Horie, N. Tanaka, Separation efficiencies in hydrophilic interaction chromatography, Journal of chromatography A, 1184 (2008) 474-503. [14] M.J. Motilva, A. Macià, A. Domínguez, A. Labrador, Liquid Chromatography Coupled to Tandem Mass Spectrometry to Analyze Imidazole Dipeptides, Imidazole Dipeptides 2015, pp. 191-213. [15] K. Shimada, K. Hirakata, Retention behavior of derivatized amino acids and dipeptides in high-performance liquid chromatography using cyclodextrin as a mobile phase additive, Journal of Liquid Chromatography & Related Technologies, 15 (1992) 1763-1771. [16] J. Zhao, D.K. Posa, V. Kumar, D. Hoetker, A. Kumar, S. Ganesan, D.W. Riggs, A. Bhatnagar, M.F. Wempe, S.P. Baba, Carnosine protects cardiac myocytes against lipid
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peroxidation products, Amino acids, 51 (2018) 123-138. [17] I. Everaert, G. Baron, S. Barbaresi, E. Gilardoni, C. Coppa, M. Carini, G. Vistoli, T. Bex, J. Stautemas, L. Blancquaert, Development and validation of a sensitive LC–MS/MS assay for the quantification of anserine in human plasma and urine and its application to pharmacokinetic study, Amino acids, 51 (2018) 103-114. [18] G. Aldini, M. Orioli, M. Carini, R. Maffei Facino, Profiling histidine-containing dipeptides in rat tissues by liquid chromatography/electrospray ionization tandem mass spectrometry,
, 39 (2004) 1417-1428.
[19] Center for Drug Evaluation and Reserch (CDER) Food and Drug Administration, Guideline for Industry Bioanalytical Method Validation, (2018). https://www.fda.gov/regulatory-information/search-fda-guidance-documents/bioanalyticalmethod-validation-guidance-industry [20] LC-MS method validation, University of Tartu, https://sisu.ut.ee/lcms_method_validation/27-identity-confirmation-examples [21] J. Drozak, M. Veiga-da-Cunha, D. Vertommen, V. Stroobant, E. Van Schaftingen, Molecular identification of carnosine synthase as ATP-grasp domain-containing protein 1 (ATPGD1), Journal of Biological Chemistry, 285 (2010) 9346-9356. [22] F. Turecek, J. Kerwin, R. Xu, K. Kramer, Distinction of N‐substituted histidines by electrospray ionization mass spectrometry, Journal of Mass Spectrometry, 33 (1998) 392-396. [23] L. Pucciarini, E. Gilardoni, F. Ianni, A. D'Amato, V. Marrone, L. Fumagalli, L. Regazzoni, G. Aldini, M. Carini, R. Sardella, Development and validation of a HPLC method for the direct separation of carnosine enantiomers and analogues in dietary supplements, Journal of Chromatography B, 1126-1127 (2019) 121747. [24] H. Abe, Role of histidine-related compounds as intracellular proton buffering constituents in vertebrate muscle, Biochemistry (Moscow), 65 (2000) 757-765.
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[25] J.E. Plowman, E.A. Close, An evaluation of a method to differentiate the species of origin of meats on the basis of the contents of anserine, balenine and carnosine in skeletal muscle, Journal of the Science of Food and Agriculture, 45 (1988) 69-78.
20
Table 1. MRM transitions of analytes and internal standard in positive ion mode method. Analytes
Carnosine
Anserine
Balenine
L-Histidyl-L-leucine (IS)
Precursor ion (m/z)
Product ion (m/z)
227
110
22
Quantifier
227
156
14
Qualifier
241
126
19
Quantifier
241
153
20
Qualifier
241
124
22
Quantifier
241
224
10
Qualifier
269
110
21
Quantifier
269
83
38
Qualifier
21
CE (eV) Comments
Table 2. The accuracy and precision of analytes. Concentration of IS (ng/mL)
Analytes
Carnosine
17.9
Anserine
Balenine
Carnosine
1,790
Anserine
Balenine
Concentration of analytes (ng/mL)
Intra-day (n = 5)
Inter-day (n = 5 × 3)
Accuracy
Precision
Accuracy
Precision
(RE, %)
(CV, %)
(RE, %)
(CV, %)
14.7
(LLOQ)
-0.18
4.83
-0.96
15.20
29.4
(low QC)
0.37
2.26
1.95
13.60
942
(mid QC)
-0.18
2.23
-1.07
13.47
11,300 (high QC)
0.00
2.93
-0.16
13.06
15.6
(LLOQ)
1.81
9.96
-0.99
15.55
31.2
(low QC)
-3.69
6.15
2.00
5.82
1,000
(mid QC)
1.81
2.55
-1.06
11.74
12,000 (high QC)
-0.07
2.44
0.01
5.12
15.6
(LLOQ)
2.66
2.81
0.59
17.13
31.2
(low QC)
-5.40
1.38
-1.21
12.98
1,000
(mid QC)
2.73
2.35
0.53
14.76
12,000 (high QC)
-0.12
3.01
-0.06
5.08
14.7
(LLOQ)
0.80
8.39
0.36
8.90
29.4
(low QC)
-1.63
1.54
-0.74
8.12
942
(mid QC)
0.80
0.90
0.35
6.27
11,300 (high QC)
-0.03
1.02
-0.01
0.95
15.6
(LLOQ)
-1.27
3.01
-2.02
5.29
31.2
(low QC)
2.56
4.92
8.97
10.29
1,000
(mid QC)
-1.25
0.98
-4.48
4.74
12,000 (high QC)
0.04
1.01
0.16
3.19
15.6
(LLOQ)
1.02
1.73
0.91
3.70
31.2
(low QC)
-2.06
2.00
-2.44
3.09
1,000
(mid QC)
1.05
0.97
1.23
4.23
12,000 (high QC)
-0.04
0.95
-0.08
4.46
22
Table 3. The relative difference of the ion ratio of meat samples to QC samples on analytes. Meat samples
Carnosine (%)
Anserine (%)
Balenine (%)
Chicken 1
-0.01
-0.15
-0.06
Chicken 2
-0.02
-0.17
0.04
Chicken 3
0.00
-0.17
0.03
Chicken 4
-0.04
-0.16
0.01
Chicken 5
0.00
-0.18
0.06
Chicken 6
-0.03
-0.17
0.12
Beef 1
-0.01
-0.12
-0.06
Beef 2
-0.02
-0.15
0.15
Beef 3
-0.02
-0.13
0.17
Beef 4
-0.02
-0.11
0.03
Beef 5
-0.03
-0.17
0.19
Beef 6
-0.01
-0.10
0.26
Sei whale (Balaenoptera borealis) meat
-0.01
-0.05
0.00
23
Table 4. The matrix effect of meat samples on analytes. Precision (CV, %)
Carnosine Anserine Balenine
Chicken (n = 6)
4.7
3.9
3.5
Beef (n = 6)
6.0
2.5
1.4
24
Table 5. The stability of analytes (n = 3). Conditions
Analytes
Nominal concentration Measured concentration (ng/mL)
Carnosine Three freeze/thaw cycles Anserine Balenine Carnosine Autosampler for 72h (15°C)
Anserine
Balenine
25
(mean ± SD, ng/mL)
Accuracy (RE, %)
29.4
27.8 ± 2.49
-5.66
11,300
11,200 ± 112
-0.20
31.2
27.4 ± 2.40
3.41
12,000
12,500 ± 159
4.07
31.2
31 ± 0.00
-1.05
12,000
12,900 ± 145
7.50
29.4
27.8 ± 1.20
-10.76
11,300
10,400 ± 346
-13.35
31.2
30.5 ± 0.48
-2.03
12,000
11,600 ± 1.36
-3.13
31.2
26.6 ± 1.20
-14.36
12,000
11,700 ± 185
-2.55
Table 6. The calibration curves of analytes. Types of calibration curve
Concentration Analytes of IS (ng/mL)
14.7 ng/mL15.1 μg/mL 15.6 ng/mLAnserine 16.0 μg/mL 15.6 ng/mLBalenine 16.0 μg/mL 14.7 ng/mLCarnosine 15.1 μg/mL 15.6 ng/mLAnserine 16.0 μg/mL 15.6 ng/mLBalenine 16.0 μg/mL Carnosine
For 100-fold dilution
For without dilution
17.9
1,790
Calibration range
26
Coefficient correlation (R2 )
Lower limit of quantification (S/N)
0.995
50.3
0.996
20.6
0.998
232.7
0.999
32.1
0.994
18.6
0.999
79.0
Table 7. Quantitative analytical results of muscle samples in previous study. Carnosine (mean ± SD)
Anserine (mean ± SD)
Balenine (mean ± SD) –
Plowman (1988)
Chicken Leg
1.242
±
0.208
2.643 ± 0.418
Abe (2000)
Chicken Leg
1.290
±
0.385
4.108 ± 0.889 0.132 ± 0.007
Aristoy (2004)
Chicken Leg
0.630
±
0.058
2.337 ± 0.187 0.101 ± 0.011
Mora (2007)
Chicken Breast
1.493
±
0.071
6.756 ± 0.333 0.055 ± 0.006
Carnegie (1983)
Beef
3.326
±
0.747
0.553 ± 0.096 0.017 ± 0.007
Plowman (1988)
Beef
3.964
±
0.803
0.615 ±
Abe (2000)
Ox Leg
5.905
±
0.837
1.427 ± 0.420 0.025 ± 0.006
Aristoy (2004)
Beef Top loin
3.725
±
0.322
0.597 ± 0.042 0.020 ± 0.001
Aristoy (2004)
Beef Neck
2.534
±
0.280
0.252 ± 0.019 0.016 ± 0.001
0.118
–
Values indicate contents (mg/g of wet meat), which were converted from μmol/g or mg/100g shown in the references. – : Not shown in the reference.
27
Figure legends Fig. 1. Chemical structures of (A) L-carnosine, (B) L-anserine, (C) L-balenine, and (D) Lhistidyl-L-leucine [internal standard (IS)].
Fig. 2. MS/MS spectra of (A) carnosine, (B) anserine, (C) balenine, and (D) L-histidyl-Lleucine [internal standard (IS)]. Arrows and asterisks indicate ions used for the quantification and qualification of the respective compounds. (E) Chemical structures of the fragment ions labeled in Fig. 2A–C.
Fig. 3. MS/MS chromatograms of (A) carnosine, (B) anserine, and (C) balenine in a mid QC sample. Black lines indicate MS/MS chromatograms for quantifications of carnosine (m/z 227 → 110), anserine (m/z 241 → 126), and balenine (m/z 241 → 124). Red lines indicate MS/MS chromatograms for qualifications of carnosine (m/z 227 → 156), anserine (m/z 241 → 153), and balenine (m/z 241 → 224). Each arrow indicates a peak produced by daughter ions of carnosine, anserine, or balenine. Asterisks of B and C indicate daughter ions produced from the parent ions of balenine and anserine, respectively.
Fig. 4. MS/MS chromatograms of L-histidyl-L-leucine [internal standard (IS)] in (A) an ISfree chicken sample, (B) an IS-free beef sample, (C) an IS-free whale meat sample, and (D) a mid QC sample containing 17.9 ng/mL of IS. MS/MS chromatograms of m/z 269 → 110 for the quantification (black line) and of m/z 269 → 83 for the qualification (red line) in IS.
28
Fig. 5. MS/MS chromatograms of (A, D) carnosine, (B, E) anserine, and (C, F) balenine in meat samples. Black lines indicate MS/MS chromatograms for quantifications of carnosine (m/z 227 → 110), anserine (m/z 241 → 126), and balenine (m/z 241 → 124). Red lines indicate MS/MS chromatograms for qualifications of carnosine (m/z 227 → 156), anserine (m/z 241 → 153), and balenine (m/z 241 → 224). (A–C) and (D–F) correspond to chicken and whale meat samples, respectively. Arrows indicate the peaks used for quantifying each imidazole dipeptide.
Fig. 6. Box and whisker plots of the contents of the imidazole dipeptides in (A) chicken, (B) beef, and (C) whale meat samples. The box bounds the interquartile range between the first quartile and the third quartile. The horizontal line within the box denotes the median. The whiskers extend to the minimum and the maximum. Plots of the same color indicate the same samples for a given type of meat.
29
30
31
32
33
34
35
Highlights 1.
A mix-mode column separated imidazole dipeptides, carnosine and its methylated analogs, without derivatization.
2.
An improved LC-ESI-MS/MS method exhibits the high selectively and low LLOQ.
3.
The validated method was successfully applied to the quantification of imidazole dipeptides in meat samples.
36
Conflicts of interest The authors declare they have no actual or potential competing conflict of interest.
37
S1570-0232(19)30830-X https://doi.org/10.1016/j.jchromb.2019.121826 CHROMB 121826
To appear in:
Journal of Chromatography B
Received Date: Revised Date: Accepted Date:
24 May 2019 6 October 2019 8 October 2019
Please cite this article as: R. Uenoyama, M. Miyazaki, T. Miyazaki, Y. Shigeno, Y. Tokairin, H. Konno, T. Yamashita, LC-ESI-MS/MS quantification of carnosine, anserine, and balenine in meat samples, Journal of Chromatography B (2019), doi: https://doi.org/10.1016/j.jchromb.2019.121826
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© 2019 Published by Elsevier B.V.
LC-ESI-MS/MS quantification of carnosine, anserine, and balenine in meat samples Reiko Uenoyama1, Masao Miyazaki1, Tamako Miyazaki1, Yuhei Shigeno2, Yoshinori Tokairin2, Hiroyuki Konno2, and Tetsuro Yamashita1
1
Department of Biological Chemistry and Food Sciences, Faculty of Agriculture, Iwate
University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan 2
Department of Biochemical Engineering, Graduate School of Science and Engineering,
Yamagata University, 4-3-16 Jounan, Yonezawa, Yamagata 992-0038, Japan
*Corresponding author. [email protected] (Tetsuro Yamashita)
1
Abstract The histidine-containing imidazole dipeptide carnosine and its methylated analogs anserine and balenine are present at high concentrations in vertebrate tissues. Although the physiological functions of the imidazole dipeptides have not been elucidated yet, it has been suggested that they play significant biological roles in animals. Despite increasing interest, few studies have challenged the quantifications of carnosine, anserine, and balenine by a single HPLC run because they have similar retention times. In this study, we developed a method to quantify these imidazole dipeptides in meat samples using an LC-ESI-MS/MS triple-quadrupole mass spectrometer. We improved the liquid chromatographic separation of the imidazole peptides by applying a mix-mode column, which provides both normal phase and ion exchange separations, and developed multiple reaction-monitoring of the transitions for quantification of m/z 227 → 110 for carnosine, m/z 241 → 126 for anserine, m/z 241 → 124 for balenine, and m/z 269 → 110 for L-histidyl-L-leucine (internal standard). The established method met all pre-defined validation criteria. Intra- and inter-day accuracy and precision were ± 10.0% and ≤ 14.8%, respectively. The ranges of quantifications were 14.7 ng/mL–1.5 mg/mL for carnosine, 15.6 ng/mL–1.6 mg/mL for anserine, and 15.6 ng/mL–1.6 mg/mL for balenine. In conclusion, the validated method was successfully applied to the quantification of imidazole dipeptides in biological samples without derivatization.
2
1. Introduction Carnosine (β-alanylhistidine, Fig. 1A) and its methylated analogs anserine (β-alanyl-1methylhistidine, Fig. 1B) and balenine (β-alanyl-3-methylhistidine, ophidine, Fig. 1C) are histidine-containing imidazole dipeptides, which are distributed in vertebrate tissues, especially in the skeletal muscle [1], the heart, and the brain [2, 3]. Although the physiological functions of these dipeptides have not been clearly elucidated [4], it has been suggested that the imidazole dipeptides function as buffers against the accumulation of lactic acid in muscle tissues [5], as inducers of enzymes expressed in muscles [6], as precursors for the synthesis of neurotransmitters [7], and as scavengers of free radicals [8]. The ratio of the imidazole dipeptides is characteristic in meat samples of different species. Anserine and balenine are present at higher concentrations (up to 10 mM) compared with other imidazole dipeptides in chicken and whale meat samples, respectively [3, 4, 9]. Unlike meat proteins that are degraded during cooking, the imidazole dipeptides are not degraded because they do not have a secondary or tertiary structure; thus, they are suitable targets for species identification [5, 9]. Quantifying the tissue contents of the imidazole dipeptides will be useful in identifying the species from which processed meats originated as an anti-counterfeiting method [5, 9]. High-performance liquid chromatography (HPLC) allows the analytical separation of the imidazole dipeptides contained in tissue samples. Reverse-phase (RP)-HPLC and hydrophilic interaction chromatography (HILIC) have been applied to separate the imidazole dipeptides in tissue samples, followed by detection using ultraviolet (UV) and fluorescence detectors [3, 10-12]. RP-HPLC is a powerful separation technique for various low molecular weight organic compounds, and results in better separation of the compounds compared with HILIC [13]. However, one disadvantage of RP-HPLC is the low retention time of polar molecules, including the imidazole dipeptides [14]. Therefore, it is necessary to derivatize the
3
dipeptides before RP-HPLC [15], although this derivatization process is time-consuming. Moreover, detection using UV spectroscopy of the imidazole dipeptides has several disadvantages, including lower selectivity and a higher value of lower limit of quantification (LLOQ) compared to the fluorescence detector. However, for fluorescence detection of the dipeptides, fluorescence labeling of the samples is necessary for HPLC sample preparations. In the last dozen years, electrospray ionization-mass spectrometry (ESI-MS) has been used as a detection method for the analysis of the imidazole dipeptides [16, 17]. Anserine and balenine, which are structural isomers, were quantified despite the subtle difference of these structures using an LC-MS system consisting of RP-HPLC and an ESI-ion trap mass spectrometer (ITMS) [18]. However, the LC-ESI-ITMS system was necessary to carry out MS/MS/MS (MS3) of anserine and balenine for discriminating them; MS/MS (MS2) of them could not adequately discriminate them. To overcome these issues, this study aimed to optimize liquid chromatography-ESItandem mass spectrometry (LC-ESI-MS/MS) for the quantification of carnosine, anserine, and balenine simultaneously in tissue samples. To improve separation of the imidazole dipeptides, we applied a mix-mode column, which provides both normal phase and ion exchange separations to HPLC. Furthermore, we used a triple-quadrupole mass spectrometer for quantifications of the imidazole dipeptides. This method shows higher selectivities and better LLOQs for the quantification of carnosine, anserine, and balenine without derivatization compared to previous methods described above.
2. Materials and Methods 2.1. Chemicals Acetonitrile (LC-MS grade, ≥ 99.9% purity) was purchased from Honeywell (Morristown, NJ, USA). Formic acid (LC/MS grade, 99% purity), 1 M ammonium formate
4
solution (HPLC grade), ethanol (reagent grade, 99.5% purity), chloroform (reagent grade, 99.0% purity), L-carnosine (≥ 98.0% purity), and L-anserine nitrate (≥ 95.0% purity) were purchased from Wako Pure Chemical Corp. (Osaka, Japan). L-Balenine (99.9% purity) was purchased from Hamari Chemicals, Ltd. (Osaka, Japan). L-Histidyl-L-leucine (His-Leu, ≥ 99.0% purity, Fig. 1D) was purchased from Peptide Institute, Inc. (Osaka, Japan).
2.2. Preparation of stock solution, calibration standards, and quality control samples Stock solutions of carnosine, anserine, and balenine were prepared in Milli-Q (MQ) water at concentrations of 2.26, 2.40, and 2.40 mg/mL, respectively. The stock solution of His-Leu (internal standard [IS]) was prepared in MQ water at a concentration of 0.67 mg/mL. Working solutions were prepared by mixing the stock solutions and diluting with 0.1 M HCl in MQ water. The stock and working solutions were stored at –20°C until analysis. The carnosine, anserine, and balenine concentrations in the calibration standard solutions were (14.7, 15.6, and 15.6 ng/mL), (58.8, 62.4, and 62.4 ng/mL), (236, 250, and 250 ng/mL), (942, 1,000, and 1,000 ng/mL), (3,770, 4,000, and 4,000 ng/mL), and (15,100, 16,000, and 16,000 ng/mL), respectively. The carnosine, anserine, and balenine concentrations were (14.7, 15.6, and 15.6 ng/mL) for the LLOQ quality control (QC) samples, (29.4, 31.2, and 31.2 ng/mL) for the low QC samples, (942, 1,000, and 1,000 ng/mL) for the medium (mid) QC samples, and (11,300, 12,000, and 12,000 ng/mL) for the high QC samples. These calibration standard solutions and QC samples were spiked with the IS at 17.9 ng/mL or 1,790 ng/mL as described below.
2.3. Meat samples Six chicken and six beef samples were obtained from Japanese supermarkets. We also used a meat sample from Sei whale (Balaenoptera borealis) caught by whaling for scientific
5
purposes during the summer season in the Northwestern Pacific in 2017 under National Oceanic and Atmospheric Administration/National Marine Fisheries Service permission.
2.4. Sample preparation An aliquot (5.0 g) of each lean meat was minced with scissors and homogenized on ice with 20 mL of ethanol spiked with 134 μg of IS. The homogenate was centrifuged at 3,000 rpm for 10 min at 4°C. The supernatant was transferred to a separating funnel. The precipitate was rehomogenized with 20 mL of 80% ethanol in MQ water and, after centrifugation, the supernatant was transferred to the separating funnel; this process was repeated twice to improve the recovery rate. Then, 60 mL of chloroform was added to the supernatant, which was shaken until only a single layer was present. After adding 40 mL of MQ water, the separating funnel was shaken for 2 min to separate the aqueous and chloroform layers. The upper aqueous layer was transferred to a recovery flask, and then, after removing ethanol by rotary evaporation, was diluted to 75 mL with HCl in MQ water (the final concentration of HCl was 0.1 M). The solution was filtered through a 0.45-μm filter (Merck Millipore, Burlington, MA, USA) and transferred into a clean autosampler vial with low peptide adsorption (PSVial100; AMR, Inc., Tokyo, Japan). The meat samples that were diluted 100fold with 0.1 M HCl in MQ water were also analyzed. Each meat sample was analyzed in three independent LC-ESI-MS/MS experiments.
2.5. LC-ESI-MS/MS This study used the LCMS-8040 system (Shimadzu Co., Kyoto, Japan). For chromatographic separation of the imidazole dipeptides, the mix-mode column (Intrada Amino Acid; 3 μm; 3 mm i.d. × 50 mm; Imtakt, Kyoto, Japan) was used at 40°C. Each sample (15 μL) was injected with an autosampler (SIC-30AC; Shimadzu Co.) that was set at
6
15°C. The needle of the autosampler was washed with MQ water. Separations were performed using the mobile phases of solvent A (0.1% formic acid in acetonitrile) and solvent B (20% acetonitrile and 80 mM ammonium acetate in MQ water) at a flow rate of 0.4 mL/min. The time program was as follows: 50% B for 14 min, 100% B for 2 min, and 50% for 5 min, for a total run time of 21 min. The effluent from the HPLC step was introduced online into a triple-quadrupole mass spectrometer. The mass spectrometer was operated in the multiple reaction-monitoring (MRM) mode using the following parameters: 100 ms of dwell time, 230 kPa of collision-induced dissociation gas, ESI in positive mode, 3 L/min of nebulizer gas, and 15 L/min of drying gas heated at 250°C. Data acquisition and processing were performed by LabSolutions (version 5.97, Shimadzu Co.).
2.6. Method validation The analytical method validation was conducted based on the bioanalytical method validation guidelines of the U.S. Food and Drug Administration [19]. The following validation parameters were verified: accuracy and precision, selectivity, matrix effect, recovery, stability, carryover, calibration model, and LLOQ. Values of accuracy and precision, calibration model, and LLOQ were calculated by LabSolutions (version 5.97, Shimadzu Co.).
2.6.1. Accuracy and precision Accuracy and precision were evaluated by analyzing five replicates of LLOQ, low, mid, and high QC samples in three independent validation runs. Intra- and inter-day accuracy values were described as the relative error [RE (%)]. The intra- and inter-day precision values were expressed as the coefficient of variation [CV (%)]. At each concentration level, the accuracy and precision were required to be within ± 15% and ≤ 15%, respectively. For the
7
LLOQ, the accuracy and precision were required to be within ± 20% and ≤ 20%, respectively.
2.6.2. Selectivity and matrix effect To investigate the selectivity of the imidazole dipeptides that are endogenous compounds in meat samples, the potential interferences of endogenous substances were evaluated in six chicken, six beef, and one whale meat samples by calculating the relative difference of the ion ratio (%) as follows [20]: The relative difference of the ion ratio =
Ion ratio in meat samples – Ion ratio in QC samples × 100 (%) Ion ratio in QC samples
, where the ion ratio was calculated as follows: Ion ratio =
Peak area of the ion for quantification Peak area of the ion for qualification
The cross-analyte/IS interference was assessed by comparing the retention time between each imidazole dipeptide and IS. The matrix effect was verified in six chicken and six beef samples. To assess matrix effects, authentic carnosine (2,260 ng), anserine (2,400 ng), and balenine (2,400 ng) were spiked in 1 mL of 100-fold-diluted meat samples. Since the imidazole dipeptides are present at high concentrations in meat samples, matrix effects were calculated as follows: Matrix effect Peak areas of each measured analyte concentration in meat samples spiked with authentic analytes – Peak areas of each measured endogenous analyte concentration in meat samples = Peak areas of each measured spiked value of the authentic analytes in 0.1 M HCl in MQ water The matrix effect was considered acceptable if the precision [CV (%)] in the six chicken and six beef samples was ≤ 15%.
2.6.3. Recovery and stability
8
The extraction recovery was evaluated by the recovery of the IS; i.e., the peak area ratio of the IS in six chicken and six beef samples prepared with 100-fold dilution which were spiked IS before extraction to in these which were spiked IS after extraction (IS concentration, 17.9 ng/mL) in triplicate. The stabilities of carnosine, anserine, and balenine were assessed after freezing and thawing fresh QC samples three times and after incubating fresh QC samples at 15°C for 72 h in the autosampler. These stability analyses were performed using low and high QC samples in triplicate. Each analyte was considered stable if its measured concentration after the treatments was within ± 15% of its measured concentration in the fresh QC samples.
2.6.4. Carryover For the assessment of carryover effects, the blank sample (0.1 M HCl in MQ water) was twice analyzed by the LC-ESI-MS/MS system immediately following analysis of an upper LOQ (ULOQ) sample spiked with the IS (final concentration, 1,790 ng/mL) in each analytical run.
2.6.5. Calibration Calibration curves were generated by calculating the peak area ratios of carnosine, anserine, and balenine to the IS versus the concentrations with a 1/concentration2 weighting factor. At least 75% of the non-zero standards (including the LLOQ and ULOQ standards) in every run were required to be ± 15% of their nominal value (± 20% for LLOQ samples). The correlation coefficient was calculated for each run.
2.6.6. LLOQ The signal-to-noise (S/N) ratio was calculated by LabSolutions software using the
9
ASTM algorithm. The LLOQ of the assay was assessed in each analytical run at an S/N ratio greater than 10.
2.7. Statistical analysis Statistical significance was tested using the non-parametric Wilcoxon matched-pair signed rank test by JMP software (ver. 12.0; SAS Institute, Cary, NC, USA).
3. Results and Discussion 3.1. MRM transitions of carnosine, anserine, and balenine Product ion scans of carnosine, anserine, and balenine were carried out to establish the MRM transitions of these analytes. Each compound was introduced directly into the ESIMS/MS using the solvent (36% acetonitrile, 0.02% formic acid, and 64 mM ammonium acetate in MQ water), and fragmentation patterns were compared among these analytes. In the MS/MS spectrum of a precursor ion of carnosine, [M+H]+ at m/z 227, major product ions were detected at m/z 210, 181, 164, 156, and 110 (Fig. 2A), as described previously [21]. The previous study reported that an ion detected at m/z 110 (Fig. 2E, a) is produced by the loss of H2O and CO from an ion with m/z 156 (Fig. 2E, b), which corresponds to a protonated histidine. Since MS/MS analysis of carnosine exhibited its highest ion at m/z 110, this ion was selected for the quantification of carnosine using the MRM method. The ion at m/z 156 was selected for the qualification of carnosine. In the MS/MS spectrum of a precursor ion of anserine, a 1-methylated analog of carnosine with [M+H]+ at m/z 241, major product ions were detected at m/z 170, 153, 126, 124, 109, 96, and 68 (Fig. 2B). On the other hand, in the MS/MS spectrum of a precursor ion of balenine, a 3-methylated analog of carnosine with [M+H]+ at m/z 241, major product ions were detected at m/z 224, 195, 178, 170, 136, 124, and 109 (Fig. 2C). Notably, the MS/MS
10
fragmentations of the product ions of anserine and balenine were different. In agreement with Turecek et al. [22], an ion detected at m/z 126 in the MS/MS spectrum of anserine (Fig. 2E, c) was produced by the loss of CO2 from an ion with m/z 170 (Fig. 2E, d), which corresponds to protonated 1-methylhistidine. By contrast, an ion detected at m/z 124 in the MS/MS spectrum of balenine (Fig. 2E, e) was produced by the loss of H2O and CO from an ion with m/z 170 (Fig. 2E, f), which corresponds to protonated 3-methylhistidine. These results suggest that the difference in methylated positions between anserine and balenine caused the difference in their MS/MS fragmentations. More specifically, in balenine, an amide proton may be easily activated by a nitrogen at position 1 of the imidazole ring since the amide proton and the imidazole nitrogen form a six-membered ring; thus, an imine would be formed by the desorption of CO2. By contrast, the amide proton of anserine would not likely be activated by the imidazole nitrogen because of steric hindrance caused by binding of the methyl group to the imidazole nitrogen; thus, an amine state is maintained, even with the loss of CO2. Since MS/MS analysis of balenine produced the highest ion at m/z 124 (Fig. 2E, e), we selected this ion for the quantification of balenine. The ion at m/z 224 was detected only from balenine, and not from anserine, hence this ion was selected for qualification of balenine. Although MS/MS analysis of anserine produced the highest ion at m/z 109, this ion was also detected as a major ion in balenine. Therefore, the other ion detected at m/z 126 was selected for the quantification of anserine. Although the ion at m/z 153 was detected only for anserine but not from balenine, its intensity was lower than that of the ion at m/z 126. Therefore, the ion detected at m/z 153 was selected for the qualification of anserine. The MRM transitions of carnosine, anserine, and balenine for quantifications and qualifications are shown in Table 1. The collision energies were optimized to generate the highest signal intensities of the ions to be quantified and qualified.
11
3.2. LC-ESI-MS/MS analyses of carnosine, anserine, and balenine The MS/MS chromatograms of the imidazole dipeptides are illustrated in Fig. 3. In the LC-ESI-MS/MS analysis of the mid QC samples, balenine (C), anserine (B), and carnosine (A) were eluted at 9.4, 10.9, and 11.6 min, respectively. The ions for the quantification of balenine (m/z 241 → 124) and anserine (m/z 241 → 126) were also detected as minor peaks at 10.9 and 9.4 min, respectively. This is because the precursor ions of balenine and anserine produced MS/MS fragment ions at m/z 126 and m/z 124, respectively. However, since balenine and anserine were not co-eluted in the HPLC system with the mix-mode column, we concluded that ions with m/z 241 → 124 and m/z 241 → 126 are useful to quantify balenine and anserine, respectively.
3.3. Internal standard selection In our work, His-Leu was selected as the IS because it is an imidazole dipeptide. In the MS/MS spectrum of the His-Leu precursor ion of [M+H]+ at m/z 269, a major product ion was detected at m/z 110 (Fig. 2D). His-Leu displayed similar chromatographic behavior as the other imidazole dipeptides, but the retention time (7.8 min) was different, indicating that there was no cross-analyte/IS interference with this method. Furthermore, the MS/MS chromatogram of m/z 269 → 110 for the IS was compared between the IS-free meat samples and the calibration/QC samples containing 17.9 ng/mL of IS (Fig. 4). No interferences of endogenous substances to the IS were confirmed based on the result that the IS responses in the IS-free meat samples were ≤ 5% of the average IS responses of the calibration and QC samples. Thus, the m/z 269 → 110 ion was selected for the quantification of His-Leu.
3.4. Method validation 3.4.1. Accuracy and precision
12
The accuracy and precision results for quantification of the imidazole dipeptides are shown in Table 2. The intra- and inter-day accuracy values of the imidazole dipeptides were within ± 5.4% and ± 9.0%, respectively, for all of the low, mid, and high QC samples. The intra- and inter-day precision values were ≤ 4.9% and ≤ 14.8%, respectively. For the LLOQ samples, the intra- and inter-day accuracy values were within ± 2.7% and ± 2.0%, respectively. The intra- and inter-day precision values were ≤ 10.0% and ≤ 17.1%, respectively. These results indicated that the established method for quantification of the imidazole dipeptides is highly accurate and precise.
3.4.2. Selectivity and matrix effect Since the imidazole dipeptides are contained as endogenous compounds in meat samples, it was difficult to evaluate their selectivity using blank matrix samples. Therefore, we evaluated the relative difference of the ion ratio of each imidazole dipeptide to establish the selectivity (Table 3). The relative difference of the imidazole dipeptides was within ± 0.26% for all of the meat samples. These results indicated that no significant interference with endogenous substances contained in meat samples was found at the retention time of each analyte. The matrix effects of six chicken and six beef samples on the imidazole dipeptide are shown in Table 4. All of the matrix effects were ≤ 6.0%, indicating that there was no matrix effect of the developed method for quantification of the imidazole dipeptides.
3.4.3. Recovery and stability The mean extraction recovery values of the IS were 87.5 ± 2.5% and 85.8 ± 9.9% in six chicken and six beef samples, respectively. The stabilities of the imidazole dipeptides are shown in Table 5. The imidazole dipeptides of the low and high QC samples were stable after
13
three freeze/thaw cycles and incubation at 15°C for 72 h.
3.4.5. Carryover No carryovers of the imidazole dipeptides and the IS were observed in the first blank samples after analyzing an ULOQ sample containing 1,790 ng/mL of the IS. Therefore, the concentrations of the imidazole dipeptides under the ULOQ were appropriate to quantify without a carryover effect.
3.4.6. Calibration and LLOQ Our preliminary experiments showed that the balenine content was approximately 80fold higher than that of anserine in the whale meat samples, and the carnosine content was approximately 160-fold higher than that of balenine in the beef samples. Therefore, to quantify the contents of each imidazole dipeptide in meat samples, we analyzed both meat samples with and without 100-fold dilution. Since the final concentrations of the IS were different between meat samples with and without the dilution, we generated two calibration curves based on the different IS concentrations. Carnosine, anserine, and balenine with low concentrations (< 1,470, < 1,560, and < 1,560 ng/mL) in meat samples were quantified without dilution using a calibration curve based on 1,790 ng/mL of the His-Leu IS. Carnosine, anserine, and balenine with high concentrations (> 1,470, > 1,560, and > 1,560 ng/mL) in meat samples were quantified with 100-fold dilution using a calibration curve based on 17.9 ng/mL of the His-Leu IS. Table 6 shows that the ranges of concentration for the two calibration curves were 14.7 ng/mL–15.1 μg/mL for carnosine and 15.6 ng/mL–16.0 μg/mL for anserine and balenine. Linearity was acceptable with the correlation coefficient (r2) exceeding 0.994 for the imidazole dipeptides.
14
In previous studies using LC-ESI-ITMS to analyze the imidazole dipeptides in meat samples, LLOQ was 12.5 pmol for each imidazole dipeptide [18]. In this study using LCESI-MS/MS to quantify the imidazole dipeptides, the LLOQs (S/N > 10:1) were 14.7 ng/mL for carnosine, 15.6 ng/mL for anserine, and 15.6 ng/mL for balenine, which convert to approximately 1 pmol injected for each imidazole dipeptide (Table 6). LC-ESI-MS/MS analysis of meat samples, using samples that were prepared at 100-fold dilution, enabled quantification of the imidazole dipeptides over the wide ranges of 14.7 ng/mL–1.5 mg/mL for carnosine, 15.6 ng/mL–1.6 mg/mL for anserine, and 15.6 ng/mL–1.6 mg/mL for balenine. These ranges convert to 0.977 nmol/g–100 μmol/g of wet mass tissue for the imidazole dipeptides, which was wider than the range of the previous reports, (15–1,000 nmol/g) [18] and (0.1–0.8 μg/mL for L-carnosine) [23]. These method validation results indicate that the developed method improves selectivity and the LLOQ of anserine, carnosine, and balenine, and provides wider concentration ranges compared with the previous researches [18, 23].
3.5. Quantification of carnosine, anserine, and balenine in meat samples Fig. 5 shows representative MS chromatograms of the imidazole dipeptides in the meat samples. For the quantifications of carnosine, anserine, and balenine, adequate separations of the imidazole dipeptides were achieved in a single run of each meat sample as well as the mid QC sample. A recent study applied a chirobiotic T column which contains the glycopeptide teicoplanin covalently bonded to a high purity spherical silica gel [23]. As compared to the chirobiotic T column, the mix-mode column providing both normal phase and ion exchange separations shows the better separation for the imidazole dipeptide. Thus, the validated method was applied to quantify the imidazole dipeptides in the chicken, beef, and whale meat samples. For quantification of the imidazole dipeptides in the meat samples, 100-fold dilution
15
was necessary to obtain values within the calibration range, except for balenine in the chicken and beef samples. Fig. 6 shows the quantification results for the imidazole dipeptides in the meat samples for the three independent LC-ESI-MS/MS runs. Good repeatability was observed for each sample. The content of each imidazole dipeptide per gram of wet meat followed a non-normal distribution in the three types of meats. Thus, the contents were evaluated using the nonparametric Wilcoxon matched-pair signed rank test. Beef and chicken samples showed significant differences among contents of the imidazole dipeptides (p<0.0001). The highest contents in chicken and beef samples corresponded to anserine and carnosine, respectively. Balenine had the lowest content in the chicken and beef samples, in contrast to a whale meat sample. Each imidazole dipeptide content was different within a given type of meat. Table 7 shows the contents of the imidazole dipeptides in meat samples reported in other studies [3, 5, 10, 24, 25]. Our quantified contents are consistent with those data. Therefore, we propose that our developed method can be used to quantify the imidazole dipeptides in meat samples.
4. Conclusions We developed a quantification method for carnosine, anserine, and balenine in meat samples using both HPLC with a mix-mode column and ESI triple-quadrupole mass spectrometer. The validated method exhibits high selectivity, low LLOQ, and wide ranges of concentrations for the simultaneous quantification of the imidazole dipeptides in biological samples. This optimized method will contribute to the anti-counterfeiting method and the elucidation of biological functions of imidazole dipeptides in vertebrates.
Conflicts of interest
16
The authors declare they have no actual or potential competing conflict of interest.
Acknowledgement We thank Ms. Manami Kobayashi of Shimadzu Co. for experimental supports.
References [1] H. Stuerenburg, The roles of carnosine in aging of skeletal muscle and in neuromuscular diseases, Biochemistry (Moscow), 65 (2000) 862-865. [2] L. Bonfanti, P. Peretto, S. De Marchis, A. Fasolo, Carnosine-related dipeptides in the mammalian brain, Progress in Neurobiology, 59 (1999) 333-353. [3] L. Mora, M.A. Sentandreu, F. Toldra, Hydrophilic chromatographic determination of carnosine, anserine, balenine, creatine, and creatinine, Journal of Agricultural and Food Chemistry, 55 (2007) 4664-4669. [4] T. Nagasawa, T. Yonekura, N. Nishizawa, D.D. Kitts, In vitro and in vivo inhibition of muscle lipid and protein oxidation by carnosine,
, 225
(2001) 29-34. [5] P.R. Carnegie, M.Z. Ilic, M.O. Etheridge, M.G. Collins, Improved high-performance liquid chromatographic method for analysis of histidine dipeptides anserine, carnosine and balenine present in fresh meat, Journal of Chromatography A, 261 (1983) 153-157. [6] P. Johnson, J.L. Hammer, Effects of L-1-methyl-histidine and the muscle dipeptides carnosine and anserine on the activities of muscle calpains, Comparative Biochemistry and Physiology. B, 94 (1989) 45-48. [7] E.A. Decker, A.D. Crum, J.T. Calvert, Differences in the antioxidant mechanism of carnosine in the presence of copper and iron, Journal of Agricultural and Food Chemistry, 40 (1992) 756-759.
17
[8] E.A. Decker, The role of phenolics, conjugated linoleic acid, carnosine, and pyrroloquinoline quinone as nonessential dietary antioxidants, Nutrition Reviews, 53 (1995) 49-58. [9] P.R. Carnegie, K.P. Hee, A.W. Bell, Ophidine (β-alanyl-L-3-methylhistidine, ‘balenine’) and other histidine dipeptides in pig muscles and tinned hams, Journal of the Science of Food and Agriculture, 33 (1982) 795-801. [10] M.C. Aristoy, F. Toldrá, Histidine dipeptides HPLC-based test for the detection of mammalian origin proteins in feeds for ruminants, Meat Science, 67 (2004) 211-217. [11] M. Dunnett, R.C. Harris, High-performance liquid chromatographic determination of imidazole dipeptides, histidine, 1-methylhistidine and 3-methylhistidine in equine and camel muscle and individual muscle fibres, Journal of Chromatography B, 688 (1997) 47-55. [12] L. Mora, A.S. Hernández-Cázares, M.-C. Aristoy, F. Toldrá, M. Reig, Hydrophilic interaction chromatography (HILIC) in the analysis of relevant quality and safety biochemical compounds in meat, poultry and processed meats, Food Analytical Methods, 4 (2011) 121-129. [13] T. Ikegami, K. Tomomatsu, H. Takubo, K. Horie, N. Tanaka, Separation efficiencies in hydrophilic interaction chromatography, Journal of chromatography A, 1184 (2008) 474-503. [14] M.J. Motilva, A. Macià, A. Domínguez, A. Labrador, Liquid Chromatography Coupled to Tandem Mass Spectrometry to Analyze Imidazole Dipeptides, Imidazole Dipeptides 2015, pp. 191-213. [15] K. Shimada, K. Hirakata, Retention behavior of derivatized amino acids and dipeptides in high-performance liquid chromatography using cyclodextrin as a mobile phase additive, Journal of Liquid Chromatography & Related Technologies, 15 (1992) 1763-1771. [16] J. Zhao, D.K. Posa, V. Kumar, D. Hoetker, A. Kumar, S. Ganesan, D.W. Riggs, A. Bhatnagar, M.F. Wempe, S.P. Baba, Carnosine protects cardiac myocytes against lipid
18
peroxidation products, Amino acids, 51 (2018) 123-138. [17] I. Everaert, G. Baron, S. Barbaresi, E. Gilardoni, C. Coppa, M. Carini, G. Vistoli, T. Bex, J. Stautemas, L. Blancquaert, Development and validation of a sensitive LC–MS/MS assay for the quantification of anserine in human plasma and urine and its application to pharmacokinetic study, Amino acids, 51 (2018) 103-114. [18] G. Aldini, M. Orioli, M. Carini, R. Maffei Facino, Profiling histidine-containing dipeptides in rat tissues by liquid chromatography/electrospray ionization tandem mass spectrometry,
, 39 (2004) 1417-1428.
[19] Center for Drug Evaluation and Reserch (CDER) Food and Drug Administration, Guideline for Industry Bioanalytical Method Validation, (2018). https://www.fda.gov/regulatory-information/search-fda-guidance-documents/bioanalyticalmethod-validation-guidance-industry [20] LC-MS method validation, University of Tartu, https://sisu.ut.ee/lcms_method_validation/27-identity-confirmation-examples [21] J. Drozak, M. Veiga-da-Cunha, D. Vertommen, V. Stroobant, E. Van Schaftingen, Molecular identification of carnosine synthase as ATP-grasp domain-containing protein 1 (ATPGD1), Journal of Biological Chemistry, 285 (2010) 9346-9356. [22] F. Turecek, J. Kerwin, R. Xu, K. Kramer, Distinction of N‐substituted histidines by electrospray ionization mass spectrometry, Journal of Mass Spectrometry, 33 (1998) 392-396. [23] L. Pucciarini, E. Gilardoni, F. Ianni, A. D'Amato, V. Marrone, L. Fumagalli, L. Regazzoni, G. Aldini, M. Carini, R. Sardella, Development and validation of a HPLC method for the direct separation of carnosine enantiomers and analogues in dietary supplements, Journal of Chromatography B, 1126-1127 (2019) 121747. [24] H. Abe, Role of histidine-related compounds as intracellular proton buffering constituents in vertebrate muscle, Biochemistry (Moscow), 65 (2000) 757-765.
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[25] J.E. Plowman, E.A. Close, An evaluation of a method to differentiate the species of origin of meats on the basis of the contents of anserine, balenine and carnosine in skeletal muscle, Journal of the Science of Food and Agriculture, 45 (1988) 69-78.
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Table 1. MRM transitions of analytes and internal standard in positive ion mode method. Analytes
Carnosine
Anserine
Balenine
L-Histidyl-L-leucine (IS)
Precursor ion (m/z)
Product ion (m/z)
227
110
22
Quantifier
227
156
14
Qualifier
241
126
19
Quantifier
241
153
20
Qualifier
241
124
22
Quantifier
241
224
10
Qualifier
269
110
21
Quantifier
269
83
38
Qualifier
21
CE (eV) Comments
Table 2. The accuracy and precision of analytes. Concentration of IS (ng/mL)
Analytes
Carnosine
17.9
Anserine
Balenine
Carnosine
1,790
Anserine
Balenine
Concentration of analytes (ng/mL)
Intra-day (n = 5)
Inter-day (n = 5 × 3)
Accuracy
Precision
Accuracy
Precision
(RE, %)
(CV, %)
(RE, %)
(CV, %)
14.7
(LLOQ)
-0.18
4.83
-0.96
15.20
29.4
(low QC)
0.37
2.26
1.95
13.60
942
(mid QC)
-0.18
2.23
-1.07
13.47
11,300 (high QC)
0.00
2.93
-0.16
13.06
15.6
(LLOQ)
1.81
9.96
-0.99
15.55
31.2
(low QC)
-3.69
6.15
2.00
5.82
1,000
(mid QC)
1.81
2.55
-1.06
11.74
12,000 (high QC)
-0.07
2.44
0.01
5.12
15.6
(LLOQ)
2.66
2.81
0.59
17.13
31.2
(low QC)
-5.40
1.38
-1.21
12.98
1,000
(mid QC)
2.73
2.35
0.53
14.76
12,000 (high QC)
-0.12
3.01
-0.06
5.08
14.7
(LLOQ)
0.80
8.39
0.36
8.90
29.4
(low QC)
-1.63
1.54
-0.74
8.12
942
(mid QC)
0.80
0.90
0.35
6.27
11,300 (high QC)
-0.03
1.02
-0.01
0.95
15.6
(LLOQ)
-1.27
3.01
-2.02
5.29
31.2
(low QC)
2.56
4.92
8.97
10.29
1,000
(mid QC)
-1.25
0.98
-4.48
4.74
12,000 (high QC)
0.04
1.01
0.16
3.19
15.6
(LLOQ)
1.02
1.73
0.91
3.70
31.2
(low QC)
-2.06
2.00
-2.44
3.09
1,000
(mid QC)
1.05
0.97
1.23
4.23
12,000 (high QC)
-0.04
0.95
-0.08
4.46
22
Table 3. The relative difference of the ion ratio of meat samples to QC samples on analytes. Meat samples
Carnosine (%)
Anserine (%)
Balenine (%)
Chicken 1
-0.01
-0.15
-0.06
Chicken 2
-0.02
-0.17
0.04
Chicken 3
0.00
-0.17
0.03
Chicken 4
-0.04
-0.16
0.01
Chicken 5
0.00
-0.18
0.06
Chicken 6
-0.03
-0.17
0.12
Beef 1
-0.01
-0.12
-0.06
Beef 2
-0.02
-0.15
0.15
Beef 3
-0.02
-0.13
0.17
Beef 4
-0.02
-0.11
0.03
Beef 5
-0.03
-0.17
0.19
Beef 6
-0.01
-0.10
0.26
Sei whale (Balaenoptera borealis) meat
-0.01
-0.05
0.00
23
Table 4. The matrix effect of meat samples on analytes. Precision (CV, %)
Carnosine Anserine Balenine
Chicken (n = 6)
4.7
3.9
3.5
Beef (n = 6)
6.0
2.5
1.4
24
Table 5. The stability of analytes (n = 3). Conditions
Analytes
Nominal concentration Measured concentration (ng/mL)
Carnosine Three freeze/thaw cycles Anserine Balenine Carnosine Autosampler for 72h (15°C)
Anserine
Balenine
25
(mean ± SD, ng/mL)
Accuracy (RE, %)
29.4
27.8 ± 2.49
-5.66
11,300
11,200 ± 112
-0.20
31.2
27.4 ± 2.40
3.41
12,000
12,500 ± 159
4.07
31.2
31 ± 0.00
-1.05
12,000
12,900 ± 145
7.50
29.4
27.8 ± 1.20
-10.76
11,300
10,400 ± 346
-13.35
31.2
30.5 ± 0.48
-2.03
12,000
11,600 ± 1.36
-3.13
31.2
26.6 ± 1.20
-14.36
12,000
11,700 ± 185
-2.55
Table 6. The calibration curves of analytes. Types of calibration curve
Concentration Analytes of IS (ng/mL)
14.7 ng/mL15.1 μg/mL 15.6 ng/mLAnserine 16.0 μg/mL 15.6 ng/mLBalenine 16.0 μg/mL 14.7 ng/mLCarnosine 15.1 μg/mL 15.6 ng/mLAnserine 16.0 μg/mL 15.6 ng/mLBalenine 16.0 μg/mL Carnosine
For 100-fold dilution
For without dilution
17.9
1,790
Calibration range
26
Coefficient correlation (R2 )
Lower limit of quantification (S/N)
0.995
50.3
0.996
20.6
0.998
232.7
0.999
32.1
0.994
18.6
0.999
79.0
Table 7. Quantitative analytical results of muscle samples in previous study. Carnosine (mean ± SD)
Anserine (mean ± SD)
Balenine (mean ± SD) –
Plowman (1988)
Chicken Leg
1.242
±
0.208
2.643 ± 0.418
Abe (2000)
Chicken Leg
1.290
±
0.385
4.108 ± 0.889 0.132 ± 0.007
Aristoy (2004)
Chicken Leg
0.630
±
0.058
2.337 ± 0.187 0.101 ± 0.011
Mora (2007)
Chicken Breast
1.493
±
0.071
6.756 ± 0.333 0.055 ± 0.006
Carnegie (1983)
Beef
3.326
±
0.747
0.553 ± 0.096 0.017 ± 0.007
Plowman (1988)
Beef
3.964
±
0.803
0.615 ±
Abe (2000)
Ox Leg
5.905
±
0.837
1.427 ± 0.420 0.025 ± 0.006
Aristoy (2004)
Beef Top loin
3.725
±
0.322
0.597 ± 0.042 0.020 ± 0.001
Aristoy (2004)
Beef Neck
2.534
±
0.280
0.252 ± 0.019 0.016 ± 0.001
0.118
–
Values indicate contents (mg/g of wet meat), which were converted from μmol/g or mg/100g shown in the references. – : Not shown in the reference.
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Figure legends Fig. 1. Chemical structures of (A) L-carnosine, (B) L-anserine, (C) L-balenine, and (D) Lhistidyl-L-leucine [internal standard (IS)].
Fig. 2. MS/MS spectra of (A) carnosine, (B) anserine, (C) balenine, and (D) L-histidyl-Lleucine [internal standard (IS)]. Arrows and asterisks indicate ions used for the quantification and qualification of the respective compounds. (E) Chemical structures of the fragment ions labeled in Fig. 2A–C.
Fig. 3. MS/MS chromatograms of (A) carnosine, (B) anserine, and (C) balenine in a mid QC sample. Black lines indicate MS/MS chromatograms for quantifications of carnosine (m/z 227 → 110), anserine (m/z 241 → 126), and balenine (m/z 241 → 124). Red lines indicate MS/MS chromatograms for qualifications of carnosine (m/z 227 → 156), anserine (m/z 241 → 153), and balenine (m/z 241 → 224). Each arrow indicates a peak produced by daughter ions of carnosine, anserine, or balenine. Asterisks of B and C indicate daughter ions produced from the parent ions of balenine and anserine, respectively.
Fig. 4. MS/MS chromatograms of L-histidyl-L-leucine [internal standard (IS)] in (A) an ISfree chicken sample, (B) an IS-free beef sample, (C) an IS-free whale meat sample, and (D) a mid QC sample containing 17.9 ng/mL of IS. MS/MS chromatograms of m/z 269 → 110 for the quantification (black line) and of m/z 269 → 83 for the qualification (red line) in IS.
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Fig. 5. MS/MS chromatograms of (A, D) carnosine, (B, E) anserine, and (C, F) balenine in meat samples. Black lines indicate MS/MS chromatograms for quantifications of carnosine (m/z 227 → 110), anserine (m/z 241 → 126), and balenine (m/z 241 → 124). Red lines indicate MS/MS chromatograms for qualifications of carnosine (m/z 227 → 156), anserine (m/z 241 → 153), and balenine (m/z 241 → 224). (A–C) and (D–F) correspond to chicken and whale meat samples, respectively. Arrows indicate the peaks used for quantifying each imidazole dipeptide.
Fig. 6. Box and whisker plots of the contents of the imidazole dipeptides in (A) chicken, (B) beef, and (C) whale meat samples. The box bounds the interquartile range between the first quartile and the third quartile. The horizontal line within the box denotes the median. The whiskers extend to the minimum and the maximum. Plots of the same color indicate the same samples for a given type of meat.
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Highlights 1.
A mix-mode column separated imidazole dipeptides, carnosine and its methylated analogs, without derivatization.
2.
An improved LC-ESI-MS/MS method exhibits the high selectively and low LLOQ.
3.
The validated method was successfully applied to the quantification of imidazole dipeptides in meat samples.
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Conflicts of interest The authors declare they have no actual or potential competing conflict of interest.
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