Journal of Chromatography B, 944 (2014) 49–54
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Development and validation of a LC–MS/MS method for homocysteine thiolactone in plasma and evaluation of its stability in plasma samples Beauty Arora a , Angayarkanni Narayanasamy b , Jayabalan Nirmal a , Nabanita Halder a , Santosh Patnaik a , Alok K. Ravi a , Thirumurthy Velpandian a,∗ a High Precision Bio-analytical Facility, Department of Ocular Pharmacology and Pharmacy, Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India b Department of Biochemistry and Cell Biology, Sankara Nethralaya, 18, College Road, Nungambakkam, Chennai 600006, TN, India
a r t i c l e
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Article history: Received 13 May 2013 Accepted 6 November 2013 Available online 14 November 2013 Keywords: Homocysteine thiolactone LC–MS/MS HILIC Plasma Validation Stability
a b s t r a c t The present study demonstrates the development and validation of a sensitive method for the quantification of homocysteine thiolactone (HCTL) in human plasma using the technique of LC–MS/MS. The gradient elution of HCTL was achieved within 5 min using ZIC HILIC column having acetonitrile with 0.1% formic acid and water with 0.1% formic acid. The method was validated for the linearity, sensitivity, accuracy, precision, recovery, matrix effect and stability. A good linearity was found within a range of 0.5–32.5 nmol/ml. Quantification was performed using multiple reaction monitoring (MRM) mode based on the molecular/fragment ion transitions for HCTL (118/56) and homatropine (276.1/142.2) as internal standard. Generally, HCTL levels in plasma were found to be highly unstable. In order to verify the stability of the HCTL levels in plasma for a longer period, the samples were extracted immediately and stored at −86 ◦ C. Using the above method it was found to be stable for a period of 1 month. The method was well applied for quantification of HCTL in plasma of healthy human volunteers. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Hyperhomocysteinemia is a well-established independent risk factor for the development of macrovascular and microvascular diseases [1]. Recent reports show that the increased homocysteine thiolactone (HCTL) levels are associated with diabetic macrovasculopathy [2]. HCTL is formed in all types of cell as a result of error-editing mettRNA synthetase when there is an excess of homocysteine (Hcy). The interaction of HCTL with proteins leads to protein homocysteinylation and loss of function [3]. Recently, vitreous levels of HCTL have been reported as an important marker of diabetic retinopathy [4]. A possible molecular mechanism underlying homocysteine toxicity involves metabolic conversion of homocysteine to HCTL during protein biosynthesis. HCTL can be detrimental because of its ability to modify proteins by forming adducts in which homocysteine is N-linked to the -amino group of protein lysine residues [5]. HCTL is expected in nanomolar quantities in plasma and other body fluids. Most of the currently available methods use HPLC with UV or fluorimeter for quantification of HCTL [5,6]. For example,
∗ Corresponding author. Tel.: +91 11 26593162; fax: +91 11 26588919. E-mail address: [email protected] (T. Velpandian). 1570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.11.011
authors [5] present a sensitive HPLC method with post column derivatization and fluorescence detection to examine the HCTL concentrations in human urine and plasma. In another study [6], separation of HCTL from human plasma has been achieved by ultrafiltration. HCTL is further purified and quantified by HPLC either on a reverse phase or a cation exchange micro-bore column. In an attempt to utilize mass spectroscopy, authors in study [7] have developed a quantitative method for the detection of homocysteine thiolactone (HCTL) in plasma by gas chromatography/mass spectrometry (GC/MS) technique combined with the negative chemical ionization (NCI). The procedure involved treating plasma samples with silica solid-phase extraction and further derivatizing them with heptafluorobutyric anhydride. The derivative was analyzed by GC/MS in NCI mode. In all the above mentioned studies, either derivaitization is carried out which is a time consuming and tedious process or complex separation process is utilized. Thus, it is need of hour to develop a simple, fast and robust technique. Therefore, developing a sensitive LC–MS/MS method may pave way to quantify the levels of HCTL from biological samples. Hence in this study, we attempt to develop and validate a sensitive method for the estimation of HCTL levels in plasma of healthy volunteers. Further studies were also conducted to understand and characterize the free HCTL levels and its stability on storage.
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2. Experimental 2.1. Chemicals and reagents l-Homocysteine thiolactone HCl (HCTL) was purchased from Sigma–Aldrich (St. Louis, MO, USA). Homatropine hydrobromide was purchased from Boehringer Ingelheim, Germany. Water used was of 18.2 M prepared by Milli-Q purification system (Millipore Corp., Bedford, MA, USA) with fresh distilled water as feed water. MS grade acetonitrile and formic acid were obtained from Merck, Germany. All other chemicals and solvents used were of analytical grade. 2.2. LC–MS/MS LC–MS/MS experiments were performed using 4000 Q-TRAP triple quadrupole, tandem mass spectrometer (AB Sciex, Foster City, CA, USA) coupled with ultra high performance liquid chromatography (UHPLC, Accela Thermo Fisher, Waltham, MA, USA) with auto-sampler and online vacuum degasser. All the parameters of tandem mass spectrometer and UHPLC were controlled by Analyst software, version 1.4.2 (AB Sciex, Foster City, CA, USA) and ChromQuest software, version 4.5 (Thermo Fisher, USA) respectively. 2.3. Optimization of MS detection and chromatographic conditions
stock with methanol:water (1:1, v/v) containing 0.1% formic acid to give a concentration of 0.2 mg/ml. For preparation of calibration standards in plasma, blank plasma was obtained from blood bank of All India Institute of Medical Sciences (AIIMS), New Delhi. Calibration standards in plasma were prepared by spiking the known amount of HCTL in plasma. The stock solution of HCTL (in plasma), thus prepared, was having a concentration as 651 nmol/ml. Serial dilutions from this stock solution was done with blank plasma so that HCTL concentrations ranged from 0.5 (LLOQ) to 32.5 (HLOQ) nmol/ml. Blank plasma was also subjected to quantification in triplicates for every assay. Extraction solvent was prepared by taking acetonitrile and water with 1% zinc sulphate in a ratio of 70:30. It contained IS at the concentration of 500 ng/ml. 20 l of either of the standard or plasma was mixed with 200 l of the extraction solvent. All the plasma standards were vortexed for 1 min and centrifuged at 7840 × g for 10 min. The clear supernatant was injected into the LC–MS/MS system. 2.5. Sample preparation Plasma samples were stored at −86 ◦ C and allowed to thaw at room temperature. Subsequently, 200 l of the extraction solvent was added to the 20 l of the plasma sample. The mixture was vortexed for 1 min followed by centrifugation at 7840 × g for 10 min. The supernatant was injected into the LC–MS/MS system. 2.6. Method validation
The analytical separation of HCTL was achieved using ZIC HILIC column (50 mm × 4.6 mm, 3.5 m; Merck, Darmstadt, Germany). Mobile phase consisted of (A) acetonitrile with 0.1% formic acid and (B) water with 0.1% formic acid. The gradient elution with a total run time of 5 min was set as follows: Initial, 70% A, decreased to 50% A in 3 min, increased to 70% A in 4 min and maintained till 5 min. The mobile phase was pumped at a flow rate of 0.5 ml/min. Divert valve is set to waste from 0 to 1.5 min, to mass spectrometer from 1.5 to 3.5 min and back to waste from 3.5 to 5 min. The column was equilibrated for 1 min between each analysis. The column and ambient temperature was maintained at 25 ± 1 ◦ C. Electrospray ionization in positive mode was applied using Turbo Ionspray source (AB Sciex, Foster City, CA, USA). Full scan mass spectra and fragment ion scan spectra of HCTL were obtained by flow infusion analysis (FIA). For optimization of mass parameters, 100 ng/ml of HCTL and homatropine [internal standard (IS)] were pumped using Harvard pump (Harvard Company, Reno, NV, USA) connected with a Hamilton syringe (Holliston, MA, USA) at the rate of 5 l/min. Quantification was performed using multiple reaction monitoring (MRM) mode based on the molecular/fragment ion transitions for HCTL (118/56) and homatropine (276.1/142.2). Source dependent parameters were optimized by FIA: gas 1 (40 psi); gas 2 (40 psi); curtain gas (10 psi); ion spray voltage (5500 V) and temperature (300 ◦ C). The dwell time for each MRM transition was set at 150 ms. Compound dependent parameters such as Declustering Potential (DP), Entrance Potential (EP), Collision Energy (CE) and Cell Exit Potential (CXP) were manually optimized for HCTL as 46.2, 3.3, 23 and 9 V, respectively. For IS, the compound dependent parameters: DP, EP, CE and CXP were manually optimized as 110, 5, 82 and 7 V, respectively.
The LC–MS/MS method was validated for the linearity, sensitivity, accuracy, precision, recovery, matrix effect and stability according to the currently accepted US Food and Drug Administration (FDA) Bioanalytical Method Validation Guidelines [8]. 2.6.1. Calibration curve, linearity and sensitivity Five concentrations of HCTL: 0.5, 2.03, 4.06, 8.13 and 32.5 nmol/ml were selected for plotting the plasma calibration curve. The linearity of the calibration curve was also calculated and a correlation coefficient (R2 ) of 0.99 or better was selected. The lower limit of quantification (LLOQ) was defined as the concentration with accuracy of 80–120% and precision (% CV) < 20%. 2.6.2. Accuracy and precision Intra and inter-day assay precisions were determined as % CV (Coefficient of Variance), and intra and inter-day assay accuracies were expressed as percentage of the theoretical concentration. Thereby, accuracy (%) = (found concentration/theoretical concentration) × 100. Intra-day assays were performed using five replicates during a period of one day while inter-day assays were performed for five consecutive days.
2.4. Calibration standard and quality control samples
2.6.3. Absolute recovery and matrix effect Absolute recovery was determined by comparing peak areas of HCTL spiked in plasma having the concentrations of 0.50, 8.13 and 32.5 nmol/ml (QC samples) to those of pure standard of corresponding concentrations. Absolute matrix effect (AME) was evaluated by comparing peak areas of post-extraction blank plasma samples spiked with HCTL (0.50, 8.13 and 32.5 nmol/ml) to those of pure standard of corresponding concentrations. Samples were analyzed with six determinations at each concentration.
The stock solution of l-homocysteine thiolactone (HCTL) was prepared as 1 mg/ml in water. Serial dilutions of the stock was done in acetonitrile:water (1:1, v/v) solution containing 0.1% formic acid to make calibration standards ranging from 0.5 to 32.5 nmol/ml. Working solution of homatropine (IS) was prepared by diluting the
2.6.4. Free HCTL quantification in plasma Free HCTL estimation in QC samples was done in ultra-filtrates using amicon centrifilters (Ultracel, Millipore) having the cutoff of 3000 Da. Sample loaded centrifilters were subjected to centrifugation at 8000 × g for 20 min. The ultrafiltrate amounting to 20 l was
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Fig. 1. Molecular and fragment ion spectrum of HCTL (118) showing the formation of all the transitions (56, 72, 90 and 100) along with their chemical structures.
subjected to quantification by adding 200 l of the extraction solvent followed by vortexing for 1 min. It was, further, centrifuged at 8000 × g for 10 min. 20 l of the supernatant was injected into the LC–MS/MS system. Blank plasma was also subjected to centrifugation and appropriate correction was made for the determination of free drug levels. For the quantification of HCTL, calibration curve was plotted using the serially diluted HCTL in water.
and subjected for the quantification. For every analysis, freshly prepared plasma calibration standards spiked with HCTL were used. For comparison, water spiked with HCTL having the concentrations of 2.05, 4.06 and 32.5 nmol/ml stored at −86 ◦ C were analyzed in triplicate on 0th, 7th, 15th and 30th day.
2.6.5. Stability 2.6.5.1. Effect of freeze thaw on the analyte in plasma. Three concentrations were selected to verify the stability of QC: 0.50, 4.06 and 32.5 nmol/ml. Stability test for HCTL in plasma was studied in triplicate after three freeze thaw cycles.
The method was validated for quantitative determination of HCTL in human plasma. Blood samples were collected from healthy human volunteers (n = 6) in EDTA containing vials. Plasma was separated from blood by centrifugation at 1960 × g for 10 min. Concentration of HCTL in unknown plasma samples was calculated using the standard calibration curve of HCTL in plasma.
2.6.5.2. Stability of analyte in plasma (short term and long term stability). Plasma spiked with HCTL in triplicates at the concentrations of 0.50, 4.06 and 32.5 nmol/ml were stored in three conditions, namely, room temp for 6 h, −86 ◦ C for 7 days and −86 ◦ C for 30 days. For every analysis, fresh calibration standards (HCTL spiked in plasma) were used. 2.6.5.3. Stability of analyte in water and the effect of antioxidants. Stability of HCTL in water and the possible influence of antioxidants on the stability of HCTL were also studied. For this purpose, HCTL in water was taken in triplicates with a concentration of 8.13 and 32.5 nmol/ml and stored at 4 ◦ C and −86 ◦ C. Similar procedure was followed after adding the antioxidant (0.1% ascorbate and 0.1% sodium metabisulphate) in the prepared solution with HCTL. The quantification of HCTL was done on 0th, 1st, 2nd, 7th and 60th day after the initiation of the study. For every analysis, fresh calibration standards were prepared with HCTL diluted in water. 2.6.5.4. Stability of extracted analyte from plasma. Plasma spiked with HCTL, having the concentrations of 2.05, 4.06 and 32.5 nmol/ml were analyzed in triplicates and subjected to the immediate extraction using the procedure stated in Section 2.5 and was stored at −86 ◦ C. It was analyzed on 0th, 7th, 15th and 30th day
2.7. Quantitative determination of HCTL in plasma
2.8. Calculation As HCTL is an inherent component detected in all plasma samples, the fresh frozen plasma obtained from the blood bank was quantified for the HCTL levels in all the experiments in triplicates and appropriate correction was made in the HCTL levels quantified in all the samples. This process included that the peak area of HCTL in blank plasma was subtracted from all the plasma standards which were spiked with HCTL and then the calibration curve was plotted. 3. Results 3.1. Method validation 3.1.1. Optimization of MS/MS fragmentation patterns for MRM quantitation Mass spectrometric parameters were optimized to achieve maximum abundance of molecular adduct and fragment ions. Full scan mass spectra and fragment ion scan spectra of HCTL were obtained by flow infusion analysis. In this study we observed the formation of product ion with the mass of 56, 72, 90 and 100 from the singly
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Fig. 2. Chromatogram shows the elution of l-homocysteine thiolactone (l-HCTL) and homatropine at 2.62 and 2.19 min respectively in plasma sample of a healthy human volunteer.
protonated HCTL ion having the mass of 118 in the positive ion mode. The product ion scan showing possible fragmentation pattern leading to the formation of all transitions is shown in Fig. 1. Subjecting the HCTL standard for LC–MS/MS, using multiple reaction monitoring (MRM) mode, the transition of 118 → 56 was found to show the highest intensity, therefore, it was selected for analytical work for further validation. Homatropine was used as an internal standard with the transition of 276.1 → 142.2. The fragmentation pattern for homatropine has been reported previously [9]. 3.1.2. Calibration curve, linearity and sensitivity The results showed the quadratic fit for HCTL with five point calibration curve in the range of 0.5–32.5 nmol/ml with regression coefficient of 0.995. LLOQ was found to be 0.5 nmol/ml and limit of detection (LOD) was 0.105 nmol/ml. A representative chromatogram showing the elution of HCTL and homatropine at 2.62 min and 2.19 min respectively in the human plasma of a healthy volunteer is shown in Fig. 2. 3.1.3. Accuracy and precision The inter-day and intra-day precision of the validated method ranged from 6.94 to 13.56 (% CV) and 6.98 to 13.37 (% CV) respectively. The inter-day and intra-day accuracies ranged from 96.79 to 106.61% and 98.94 to 111.54% respectively. Table 1 shows the inter-day accuracy and intra-day accuracy and precision of the QC samples. 3.1.4. Recovery and matrix effect The AME of the proposed extraction method was 84.76%, 86.23% and 87.58% for the HCTL concentration of 0.50, 8.13 and 32.5 nmol/ml, respectively. The absolute recovery of the extraction
method was 10.45%, 9.89% and 9.68% for the HCTL concentration of 0.50, 8.13 and 32.5 nmol/ml, respectively. 3.1.5. Plasma unbound free HCTL quantification In this method, we found the free HCTL concentration of the plasma samples as 11.30, 10.83 and 13.58% for 0.50, 8.13 and 32.5 nmol/ml of HCTL. The mean free HCTL concentration in the plasma samples is 11.9% which is correlating well with the absolute recovery, thus indicating that HCTL quantified in plasma is in the free form. Simultaneously, the unbound HCTL levels in ultrafiltrate were compared with the HCTL levels found in spiked plasma. This comparison revealed that free HCTL contributed 88.83–93.15% of the HCTL levels in spiked plasma. Thus indicating the presence of 90.56% unbound HCTL in plasma as a major fraction obtained by the process of extraction followed in this study. This study has revealed that although HCTL binds to plasma protein but the bound form of HCTL is not released (using this extraction solvent) and only free drug levels can be estimated. In this study, free drug levels were estimated in order to understand the less recovery obtained by the proposed method. HCTL plasma binding gave us a clue that it might be a reason for the same as free drug level was found to be very low as 11.9%. Using 1% ZnSO4 with acetonitrile is effective for clear supernatants [10] by precipitating proteins of the plasma. Considering the high plasma protein binding of HCTL, the above mixture was used as the extraction solvent in a ratio of acetonitrile and water with 1% ZnSO4 as 7:3. However, even after using this method, in this study we could not separate the protein (of plasma) bound form of HCTL. 3.1.6. Stability Studies performed to determine the drug stability in plasma indicated that HCTL was found to be unstable. The data of freeze
Table 1 Accuracy and precision of HCTL (in plasma) for the intra-day and inter-day. Theoretical concentration (nmol/ml)
0.50 2.03 4.06 8.13 32.5
Accuracy (%)
Precision (%CV)
Intra-day
Inter-day
Intra-day
Inter-day
111.54 107.48 102.17 98.94 100.03
106.61 104.80 98.69 96.79 100.13
11.30 11.88 11.66 13.37 6.98
10.95 11.88 11.17 13.56 6.94
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Table 2 Stability of HCTL in plasma at different temperatures. Nominal concentration (nmol/ml)
Freeze thaw (mean ± SD)
Short terma (mean ± SD)
Long termb (mean ± SD)
Long termc (mean ± SD)
0.50 4.06 32.5
0.5 ± 0.00 4.06 ± 0.00 32.51 ± 0.01
0.5 ± 0.28 4.06 ± 0.56 32.52 ± 2.75
0.13 ± 0.02 0.44 ± 0.06 6.91 ± 2.43
0.02 ± 0.00 0.04 ± 0.02 0.33 ± 0.03
a b c
After 6 h at room temperature. After 7 days at −86 ◦ C. After 30 days at −86 ◦ C.
Table 3 Stability of HCTL in water at two different concentrations (Conc 1: 8.13 nmol/ml and Conc 2: 32.5 nmol/ml) over the period of two months. Each value indicates mean ± SD (% stability) of concentration. No. of days
Kept at 4 ◦ C
Kept at −86 ◦ C
Conc 1 Day 0 Day 1 Day 2 Day 7 Day 60
8.15 7.91 8.83 8.45 2.20
± ± ± ± ±
Conc 2 0.17 (100%) 0.78 (97%) 0.25 (109%) 0.37 (104%) 0.07 (27%)
32.5 29.83 34.25 37.77 13.20
± ± ± ± ±
Conc 1 2.35 (100%) 2.01 (92%) 0.85 (105%) 0.67 (116%) 0.1 (41%)
8.15 7.92 8.98 8.00 6.94
± ± ± ± ±
Conc 2 0.17 (100%) 0.09 (97%) 0.38 (110%) 0.15 (98%) 0.11 (85%)
32.50 28.83 34.3 32.4 28.33
± ± ± ± ±
2.35 (100%) 1.29 (89%) 2.30 (106%) 1.06 (100%) 0.47 (87%)
Table 4 Stability of extracted HCTL in plasma over the period of one month. Nominal concentration (nmol/ml)
Day 0 (mean ± SD)
Long terma (mean ± SD)
Long termb (mean ± SD)
Long termc (mean ± SD)
2.03 4.06 32.5
2.32 ± 0.23 4.31 ± 0.22 32.60 ± 1.27
1.76 ± 0.09 3.67 ± 0.37 19.70 ± 0.70
2.28 ± 0.15 3.93 ± 0.28 23.40 ± 0.35
1.96 ± 0.10 3.45 ± 0.06 21.60 ± 0.95
a b c
After 7 days at −86 ◦ C. After 15 days at −86 ◦ C. After 30 days at −86 ◦ C.
thaw, short term and long term stability of the QC samples are shown in Table 2. Within a period of 7 days, approximately 81% of the drug was found to disappear and over 30 days, 98% of the drug degraded in QC samples. The stability of HCTL in water for two concentrations (8.13 and 32.5 nmol/ml) on 0th, 1st, 2nd, 7th and 60th day at 4 ◦ C and −86 ◦ C is shown in Table 3. Although HCTL in water was found to be stable up to 7 days both at 4 ◦ C and −86 ◦ C, HCTL degradation was observed on 60th day. The antioxidants, 0.1% ascorbate and 0.1% sodium metabisulphite could not control the deterioration of HCTL observed in water after prolong storage. Stability of the extracted HCTL in plasma at −86 ◦ C with concentrations of 2.05, 4.06 and 32.5 nmol/ml on 0th, 7th, 15th and 30th day is shown in Table 4. The results of HCTL (2.05, 4.06 and 32.5 nmol/ml) spiked in water, stored at −86 ◦ C and analyzed on 0th, 7th, 15th and 30th day showed that HCTL is stable in water for all the three concentrations up to 30 days. 3.2. Quantitative determination of HCTL in plasma HCTL was estimated in all human plasma samples and the concentration for each of them is shown in Table 5.
Table 5 Homocysteine thiolactone concentration in healthy human volunteers. S. no. (control sample)
Age/gender
HCTL conc. (nmol/ml)
1 2 3 4 5 6
30/M 43/F 30/M 24/M 36/M 33/F
0.19 0.20 0.16 0.17 0.25 0.11
4. Discussion Homocysteine thiolactone (HCTL) was discovered by serendipity in 1934 as a bi product of the digestion of methionine with hydroiodic acid, a procedure used for the determination of protein methionine [11]. In all cell types, HCTL is a product of homocysteine metabolism by methionyl-t-RNA synthetase (MetRS) [6]. In general homocysteine incorporation into the protein has been recognized as post translational, reflecting facile homocysteinylation of protein lysine residues by HCTL. The homocysteinylation leads to protein damage and has been implicated in neurodegenerative disorders, cardiovascular diseases, diabetic complications, etc. [5]. Therefore, understanding the levels of HCTL in plasma and other body fluids has been considered as an essential aspect to understand their further involvement in various disease conditions. In human serum, about half of the exogenous HCTL incorporated into protein has been reported to be released as free homocysteine after reduction with DTT [12]. As homocysteine thiolactone has been shown to react with free amino groups in proteins to form isopeptide bonds, in lysine residues [13], conventionally the total homocysteine levels were considered as an indicator by liberating the Hcy from its bound form with proteins. Considering the aliphatic nature of Hcy, the reduced Hcy was cyclized to HCTL by heating it with 6 M HCl for 30 min at 100 ◦ C. The converted HCTL was estimated by HPLC using UV at 240 nm [6]. The same research group quantified the plasma HCTL levels from its ultrafiltrate after adsorbing it in DPBS-washed charcoal, eluted by dilute acid, lyophilized and estimated using HPLC. Moreover, the results of their control experiments revealed that HCTL is stable in human plasma frozen at −86 ◦ C for at least 6 month’s time. However, its instability in human serum or plasma was explained by two reactions; enzymatic hydrolysis by homocysteine thiolactonase (paraoxonase), a calcium dependant enzyme tightly associated with HDL and
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non-enzymatic reactions with side chain amino groups of protein lysine residues [6]. In contrast to the controlled studies, we found that plasma samples spiked with HCTL were unstable even at −86 ◦ C after 7 days. We also found the disappearance of exogenously added HCTL in plasma (at the level of 32 nmol/ml) to the extent of 79% on 7th day and 99% on 30th day when the samples were kept at −86 ◦ C. Further, HCTL standards stored in water at 4 ◦ C and −86 ◦ C were compared. This comparison revealed that HCTL standards in water were stable up to 7 days at 4 ◦ C while the stability at −86 ◦ C was compromised at a period of 2 months (deteriorated beyond 10%) (Table 3). The deterioration of HCTL in water may be due to oxidation; therefore, an attempt was made with sodium metabisulphite or ascorbic acid to stabilize the same. However, in the present study we found antioxidants do not have any role in controlling the deterioration of HCTL observed in water when stored for prolonged period. In order to improve the stability of HCTL in plasma during its storage, the extraction method was slightly modified and extraction was done within 6 h from the harvesting time of plasma. Further, the stability of extracted HCTL (plasma) was conducted for a period of one month and compared with HCTL stored in water at −86 ◦ C. These experiments concluded that the new method of HCTL preservation in plasma, i.e., immediate extraction and storage at −86 ◦ C, is capable of preventing its disappearance in plasma. In this study, we used the MRM transition of 118 → 56 for the quantification of HCTL. However, other transitions such as 118 → 90 and 118 → 72 can also be combined to further improve its sensitivity in the MRM quantization. The fragmentation pattern of precursor ion leading to the formation of product ion with mass of 56 is shown in Fig. 1. It is a self charged quaternary ammonium fragment of HCTL after ring opening reaction occurred in the collision cell. The signal produced by the self-charged quaternary ammonium compounds is justifiable in the ESI-MS/MS experiments as reported in literature [14]. Enhanced HCTL synthesis is reported in human breast cancer, cystathionine synthase deficient cells or folate deficient cells [3,15,16]. Due to its ability to damage the proteins [17], HCTL has been expected to play a role in cardiovascular diseases also [3,15,18]. In our previous studies [4], we have used C 18 column (Chromolith Speed rod, Merck, Germany) for the separation of HCTL. Although the method was good enough to quantify HCTL from human vitreous humor (having trace amount of proteins), further improvement was felt to be important so as to understand its interaction with major amount of plasma proteins to determine the plasma HCTL concentrations. Due to lack of the desired sensitivity, the method was later modified to utilize hydrophilic HILIC technology for the elution of HCTL and validated. HILIC separation showed high sensitivity in the optimized analytical method. In this study, the blank plasma showed a small peak
of HCTL which is present endogenously and was quantified as 0.062 nmol/ml. Interestingly, this study has also observed a well resolved endogenous peak suspected to be d-HCTL while using HILIC separation. This peak was unresolved while reverse phase column (Chromolith Speed rod) was used. Moreover, this peak was undetectable in the standards and was only observed in spiked blood samples. Although the presence of d-amino acids are reported along with aging process [19], the relevance of our finding on the presence of d-HCTL in the plasma is yet to be ascertained. 5. Conclusion A rapid and sensitive LC–MS/MS method has been developed for the quantification of HCTL in human plasma. The method has a short run time and easy sample preparation. Moreover, this study found that collected plasma samples, if extracted immediately and stored at −86 ◦ C remain stable up to a period of one month. The assay has been successfully applied to estimate the HCTL levels in healthy human volunteers. Acknowledgement The authors would like to thank DST-FIST for providing High Precision Bio-analytical Facility (HPBAF) for this analysis. References [1] R. Clarke, L. Daly, K. Robinson, E. Naughten, S. Cahalane, B. Fowler, I. Graham, N. Engl. J. Med. 324 (1991) 1149–1155. [2] W. Gu, J. Lu, G. Yang, J. Dou, Y. Mu, J. Meng, C. Pan, Adv. Ther. 25 (2008) 914–924. [3] H. Jakubowski, L. Zhang, A. Bardeguez, A. Aviv, Circ. Res. 87 (2000) 45–51. [4] S. Barathi, N. Angayarkanni, A. Pasupathi, S.K. Natarajan, R. Pukraj, M. Dhupper, T. Velpandian, C. Muralidharan, M. Sivashanmugham, Diabetes Care 33 (2010) 2031–2037. [5] G. Chwatko, H. Jakubowski, Clin. Chem. 51 (2005) 408–415. [6] H. Jakubowski, Anal. Biochem. 308 (2002) 112–119. [7] P. Daneshvar, M. Yazdanpanah, C. Cuthbert, D.E. Cole, Rapid Commun. Mass Spectrom. 17 (2003) 358–362. [8] FDA Guidance for Industry, Bioanalytical Method Validation, 2001. [9] J. Nirmal, T. Velpandian, S.B. Singh, N.R. Biswas, V. Thavaraj, R. Azad, S. Ghose, J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 879 (2011) 585–590. [10] S. Lam, L. Boselli, Biomed. Chromatogr. 1 (1986) 177–179. [11] H.D. Baernstein, J. Biol. Chem. 106 (1934) 451–456. [12] H. Jakubowski, J. Physiol. Pharmacol. 59 (Suppl. 9) (2008) 155–167. [13] J. Perla-Kajan, T. Twardowski, H. Jakubowski, Amino Acids 32 (2007) 561–572. [14] T. Velpandian, J. Nirmal, B. Arora, A.K. Ravi, A. Kotnala, Anal. Lett. 45 (2012) 2367–2376. [15] H. Jakubowski, J. Biol. Chem. 272 (1997) 1935–1942. [16] H. Jakubowski, in: R. Carmel, D.W. Jacobsen (Eds.), Homocysteine in Health and Disease, Cambridge University Press, UK, 2001, pp. 21–31. [17] H. Jakubowski, FASEB J. 13 (1999) 2277–2283. [18] H. Jakubowski, Biomed. Pharmacother. 55 (2001) 443–447. [19] Y. Nagata, T. Akino, K. Ohno, Y. Kataoka, T. Ueda, T. Sakurai, K. Shiroshita, T. Yasuda, Clin. Sci. (Lond.) 73 (1987) 105–108.
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Development and validation of a LC–MS/MS method for homocysteine thiolactone in plasma and evaluation of its stability in plasma samples Beauty Arora a , Angayarkanni Narayanasamy b , Jayabalan Nirmal a , Nabanita Halder a , Santosh Patnaik a , Alok K. Ravi a , Thirumurthy Velpandian a,∗ a High Precision Bio-analytical Facility, Department of Ocular Pharmacology and Pharmacy, Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India b Department of Biochemistry and Cell Biology, Sankara Nethralaya, 18, College Road, Nungambakkam, Chennai 600006, TN, India
a r t i c l e
i n f o
Article history: Received 13 May 2013 Accepted 6 November 2013 Available online 14 November 2013 Keywords: Homocysteine thiolactone LC–MS/MS HILIC Plasma Validation Stability
a b s t r a c t The present study demonstrates the development and validation of a sensitive method for the quantification of homocysteine thiolactone (HCTL) in human plasma using the technique of LC–MS/MS. The gradient elution of HCTL was achieved within 5 min using ZIC HILIC column having acetonitrile with 0.1% formic acid and water with 0.1% formic acid. The method was validated for the linearity, sensitivity, accuracy, precision, recovery, matrix effect and stability. A good linearity was found within a range of 0.5–32.5 nmol/ml. Quantification was performed using multiple reaction monitoring (MRM) mode based on the molecular/fragment ion transitions for HCTL (118/56) and homatropine (276.1/142.2) as internal standard. Generally, HCTL levels in plasma were found to be highly unstable. In order to verify the stability of the HCTL levels in plasma for a longer period, the samples were extracted immediately and stored at −86 ◦ C. Using the above method it was found to be stable for a period of 1 month. The method was well applied for quantification of HCTL in plasma of healthy human volunteers. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Hyperhomocysteinemia is a well-established independent risk factor for the development of macrovascular and microvascular diseases [1]. Recent reports show that the increased homocysteine thiolactone (HCTL) levels are associated with diabetic macrovasculopathy [2]. HCTL is formed in all types of cell as a result of error-editing mettRNA synthetase when there is an excess of homocysteine (Hcy). The interaction of HCTL with proteins leads to protein homocysteinylation and loss of function [3]. Recently, vitreous levels of HCTL have been reported as an important marker of diabetic retinopathy [4]. A possible molecular mechanism underlying homocysteine toxicity involves metabolic conversion of homocysteine to HCTL during protein biosynthesis. HCTL can be detrimental because of its ability to modify proteins by forming adducts in which homocysteine is N-linked to the -amino group of protein lysine residues [5]. HCTL is expected in nanomolar quantities in plasma and other body fluids. Most of the currently available methods use HPLC with UV or fluorimeter for quantification of HCTL [5,6]. For example,
∗ Corresponding author. Tel.: +91 11 26593162; fax: +91 11 26588919. E-mail address: [email protected] (T. Velpandian). 1570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.11.011
authors [5] present a sensitive HPLC method with post column derivatization and fluorescence detection to examine the HCTL concentrations in human urine and plasma. In another study [6], separation of HCTL from human plasma has been achieved by ultrafiltration. HCTL is further purified and quantified by HPLC either on a reverse phase or a cation exchange micro-bore column. In an attempt to utilize mass spectroscopy, authors in study [7] have developed a quantitative method for the detection of homocysteine thiolactone (HCTL) in plasma by gas chromatography/mass spectrometry (GC/MS) technique combined with the negative chemical ionization (NCI). The procedure involved treating plasma samples with silica solid-phase extraction and further derivatizing them with heptafluorobutyric anhydride. The derivative was analyzed by GC/MS in NCI mode. In all the above mentioned studies, either derivaitization is carried out which is a time consuming and tedious process or complex separation process is utilized. Thus, it is need of hour to develop a simple, fast and robust technique. Therefore, developing a sensitive LC–MS/MS method may pave way to quantify the levels of HCTL from biological samples. Hence in this study, we attempt to develop and validate a sensitive method for the estimation of HCTL levels in plasma of healthy volunteers. Further studies were also conducted to understand and characterize the free HCTL levels and its stability on storage.
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2. Experimental 2.1. Chemicals and reagents l-Homocysteine thiolactone HCl (HCTL) was purchased from Sigma–Aldrich (St. Louis, MO, USA). Homatropine hydrobromide was purchased from Boehringer Ingelheim, Germany. Water used was of 18.2 M prepared by Milli-Q purification system (Millipore Corp., Bedford, MA, USA) with fresh distilled water as feed water. MS grade acetonitrile and formic acid were obtained from Merck, Germany. All other chemicals and solvents used were of analytical grade. 2.2. LC–MS/MS LC–MS/MS experiments were performed using 4000 Q-TRAP triple quadrupole, tandem mass spectrometer (AB Sciex, Foster City, CA, USA) coupled with ultra high performance liquid chromatography (UHPLC, Accela Thermo Fisher, Waltham, MA, USA) with auto-sampler and online vacuum degasser. All the parameters of tandem mass spectrometer and UHPLC were controlled by Analyst software, version 1.4.2 (AB Sciex, Foster City, CA, USA) and ChromQuest software, version 4.5 (Thermo Fisher, USA) respectively. 2.3. Optimization of MS detection and chromatographic conditions
stock with methanol:water (1:1, v/v) containing 0.1% formic acid to give a concentration of 0.2 mg/ml. For preparation of calibration standards in plasma, blank plasma was obtained from blood bank of All India Institute of Medical Sciences (AIIMS), New Delhi. Calibration standards in plasma were prepared by spiking the known amount of HCTL in plasma. The stock solution of HCTL (in plasma), thus prepared, was having a concentration as 651 nmol/ml. Serial dilutions from this stock solution was done with blank plasma so that HCTL concentrations ranged from 0.5 (LLOQ) to 32.5 (HLOQ) nmol/ml. Blank plasma was also subjected to quantification in triplicates for every assay. Extraction solvent was prepared by taking acetonitrile and water with 1% zinc sulphate in a ratio of 70:30. It contained IS at the concentration of 500 ng/ml. 20 l of either of the standard or plasma was mixed with 200 l of the extraction solvent. All the plasma standards were vortexed for 1 min and centrifuged at 7840 × g for 10 min. The clear supernatant was injected into the LC–MS/MS system. 2.5. Sample preparation Plasma samples were stored at −86 ◦ C and allowed to thaw at room temperature. Subsequently, 200 l of the extraction solvent was added to the 20 l of the plasma sample. The mixture was vortexed for 1 min followed by centrifugation at 7840 × g for 10 min. The supernatant was injected into the LC–MS/MS system. 2.6. Method validation
The analytical separation of HCTL was achieved using ZIC HILIC column (50 mm × 4.6 mm, 3.5 m; Merck, Darmstadt, Germany). Mobile phase consisted of (A) acetonitrile with 0.1% formic acid and (B) water with 0.1% formic acid. The gradient elution with a total run time of 5 min was set as follows: Initial, 70% A, decreased to 50% A in 3 min, increased to 70% A in 4 min and maintained till 5 min. The mobile phase was pumped at a flow rate of 0.5 ml/min. Divert valve is set to waste from 0 to 1.5 min, to mass spectrometer from 1.5 to 3.5 min and back to waste from 3.5 to 5 min. The column was equilibrated for 1 min between each analysis. The column and ambient temperature was maintained at 25 ± 1 ◦ C. Electrospray ionization in positive mode was applied using Turbo Ionspray source (AB Sciex, Foster City, CA, USA). Full scan mass spectra and fragment ion scan spectra of HCTL were obtained by flow infusion analysis (FIA). For optimization of mass parameters, 100 ng/ml of HCTL and homatropine [internal standard (IS)] were pumped using Harvard pump (Harvard Company, Reno, NV, USA) connected with a Hamilton syringe (Holliston, MA, USA) at the rate of 5 l/min. Quantification was performed using multiple reaction monitoring (MRM) mode based on the molecular/fragment ion transitions for HCTL (118/56) and homatropine (276.1/142.2). Source dependent parameters were optimized by FIA: gas 1 (40 psi); gas 2 (40 psi); curtain gas (10 psi); ion spray voltage (5500 V) and temperature (300 ◦ C). The dwell time for each MRM transition was set at 150 ms. Compound dependent parameters such as Declustering Potential (DP), Entrance Potential (EP), Collision Energy (CE) and Cell Exit Potential (CXP) were manually optimized for HCTL as 46.2, 3.3, 23 and 9 V, respectively. For IS, the compound dependent parameters: DP, EP, CE and CXP were manually optimized as 110, 5, 82 and 7 V, respectively.
The LC–MS/MS method was validated for the linearity, sensitivity, accuracy, precision, recovery, matrix effect and stability according to the currently accepted US Food and Drug Administration (FDA) Bioanalytical Method Validation Guidelines [8]. 2.6.1. Calibration curve, linearity and sensitivity Five concentrations of HCTL: 0.5, 2.03, 4.06, 8.13 and 32.5 nmol/ml were selected for plotting the plasma calibration curve. The linearity of the calibration curve was also calculated and a correlation coefficient (R2 ) of 0.99 or better was selected. The lower limit of quantification (LLOQ) was defined as the concentration with accuracy of 80–120% and precision (% CV) < 20%. 2.6.2. Accuracy and precision Intra and inter-day assay precisions were determined as % CV (Coefficient of Variance), and intra and inter-day assay accuracies were expressed as percentage of the theoretical concentration. Thereby, accuracy (%) = (found concentration/theoretical concentration) × 100. Intra-day assays were performed using five replicates during a period of one day while inter-day assays were performed for five consecutive days.
2.4. Calibration standard and quality control samples
2.6.3. Absolute recovery and matrix effect Absolute recovery was determined by comparing peak areas of HCTL spiked in plasma having the concentrations of 0.50, 8.13 and 32.5 nmol/ml (QC samples) to those of pure standard of corresponding concentrations. Absolute matrix effect (AME) was evaluated by comparing peak areas of post-extraction blank plasma samples spiked with HCTL (0.50, 8.13 and 32.5 nmol/ml) to those of pure standard of corresponding concentrations. Samples were analyzed with six determinations at each concentration.
The stock solution of l-homocysteine thiolactone (HCTL) was prepared as 1 mg/ml in water. Serial dilutions of the stock was done in acetonitrile:water (1:1, v/v) solution containing 0.1% formic acid to make calibration standards ranging from 0.5 to 32.5 nmol/ml. Working solution of homatropine (IS) was prepared by diluting the
2.6.4. Free HCTL quantification in plasma Free HCTL estimation in QC samples was done in ultra-filtrates using amicon centrifilters (Ultracel, Millipore) having the cutoff of 3000 Da. Sample loaded centrifilters were subjected to centrifugation at 8000 × g for 20 min. The ultrafiltrate amounting to 20 l was
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Fig. 1. Molecular and fragment ion spectrum of HCTL (118) showing the formation of all the transitions (56, 72, 90 and 100) along with their chemical structures.
subjected to quantification by adding 200 l of the extraction solvent followed by vortexing for 1 min. It was, further, centrifuged at 8000 × g for 10 min. 20 l of the supernatant was injected into the LC–MS/MS system. Blank plasma was also subjected to centrifugation and appropriate correction was made for the determination of free drug levels. For the quantification of HCTL, calibration curve was plotted using the serially diluted HCTL in water.
and subjected for the quantification. For every analysis, freshly prepared plasma calibration standards spiked with HCTL were used. For comparison, water spiked with HCTL having the concentrations of 2.05, 4.06 and 32.5 nmol/ml stored at −86 ◦ C were analyzed in triplicate on 0th, 7th, 15th and 30th day.
2.6.5. Stability 2.6.5.1. Effect of freeze thaw on the analyte in plasma. Three concentrations were selected to verify the stability of QC: 0.50, 4.06 and 32.5 nmol/ml. Stability test for HCTL in plasma was studied in triplicate after three freeze thaw cycles.
The method was validated for quantitative determination of HCTL in human plasma. Blood samples were collected from healthy human volunteers (n = 6) in EDTA containing vials. Plasma was separated from blood by centrifugation at 1960 × g for 10 min. Concentration of HCTL in unknown plasma samples was calculated using the standard calibration curve of HCTL in plasma.
2.6.5.2. Stability of analyte in plasma (short term and long term stability). Plasma spiked with HCTL in triplicates at the concentrations of 0.50, 4.06 and 32.5 nmol/ml were stored in three conditions, namely, room temp for 6 h, −86 ◦ C for 7 days and −86 ◦ C for 30 days. For every analysis, fresh calibration standards (HCTL spiked in plasma) were used. 2.6.5.3. Stability of analyte in water and the effect of antioxidants. Stability of HCTL in water and the possible influence of antioxidants on the stability of HCTL were also studied. For this purpose, HCTL in water was taken in triplicates with a concentration of 8.13 and 32.5 nmol/ml and stored at 4 ◦ C and −86 ◦ C. Similar procedure was followed after adding the antioxidant (0.1% ascorbate and 0.1% sodium metabisulphate) in the prepared solution with HCTL. The quantification of HCTL was done on 0th, 1st, 2nd, 7th and 60th day after the initiation of the study. For every analysis, fresh calibration standards were prepared with HCTL diluted in water. 2.6.5.4. Stability of extracted analyte from plasma. Plasma spiked with HCTL, having the concentrations of 2.05, 4.06 and 32.5 nmol/ml were analyzed in triplicates and subjected to the immediate extraction using the procedure stated in Section 2.5 and was stored at −86 ◦ C. It was analyzed on 0th, 7th, 15th and 30th day
2.7. Quantitative determination of HCTL in plasma
2.8. Calculation As HCTL is an inherent component detected in all plasma samples, the fresh frozen plasma obtained from the blood bank was quantified for the HCTL levels in all the experiments in triplicates and appropriate correction was made in the HCTL levels quantified in all the samples. This process included that the peak area of HCTL in blank plasma was subtracted from all the plasma standards which were spiked with HCTL and then the calibration curve was plotted. 3. Results 3.1. Method validation 3.1.1. Optimization of MS/MS fragmentation patterns for MRM quantitation Mass spectrometric parameters were optimized to achieve maximum abundance of molecular adduct and fragment ions. Full scan mass spectra and fragment ion scan spectra of HCTL were obtained by flow infusion analysis. In this study we observed the formation of product ion with the mass of 56, 72, 90 and 100 from the singly
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Fig. 2. Chromatogram shows the elution of l-homocysteine thiolactone (l-HCTL) and homatropine at 2.62 and 2.19 min respectively in plasma sample of a healthy human volunteer.
protonated HCTL ion having the mass of 118 in the positive ion mode. The product ion scan showing possible fragmentation pattern leading to the formation of all transitions is shown in Fig. 1. Subjecting the HCTL standard for LC–MS/MS, using multiple reaction monitoring (MRM) mode, the transition of 118 → 56 was found to show the highest intensity, therefore, it was selected for analytical work for further validation. Homatropine was used as an internal standard with the transition of 276.1 → 142.2. The fragmentation pattern for homatropine has been reported previously [9]. 3.1.2. Calibration curve, linearity and sensitivity The results showed the quadratic fit for HCTL with five point calibration curve in the range of 0.5–32.5 nmol/ml with regression coefficient of 0.995. LLOQ was found to be 0.5 nmol/ml and limit of detection (LOD) was 0.105 nmol/ml. A representative chromatogram showing the elution of HCTL and homatropine at 2.62 min and 2.19 min respectively in the human plasma of a healthy volunteer is shown in Fig. 2. 3.1.3. Accuracy and precision The inter-day and intra-day precision of the validated method ranged from 6.94 to 13.56 (% CV) and 6.98 to 13.37 (% CV) respectively. The inter-day and intra-day accuracies ranged from 96.79 to 106.61% and 98.94 to 111.54% respectively. Table 1 shows the inter-day accuracy and intra-day accuracy and precision of the QC samples. 3.1.4. Recovery and matrix effect The AME of the proposed extraction method was 84.76%, 86.23% and 87.58% for the HCTL concentration of 0.50, 8.13 and 32.5 nmol/ml, respectively. The absolute recovery of the extraction
method was 10.45%, 9.89% and 9.68% for the HCTL concentration of 0.50, 8.13 and 32.5 nmol/ml, respectively. 3.1.5. Plasma unbound free HCTL quantification In this method, we found the free HCTL concentration of the plasma samples as 11.30, 10.83 and 13.58% for 0.50, 8.13 and 32.5 nmol/ml of HCTL. The mean free HCTL concentration in the plasma samples is 11.9% which is correlating well with the absolute recovery, thus indicating that HCTL quantified in plasma is in the free form. Simultaneously, the unbound HCTL levels in ultrafiltrate were compared with the HCTL levels found in spiked plasma. This comparison revealed that free HCTL contributed 88.83–93.15% of the HCTL levels in spiked plasma. Thus indicating the presence of 90.56% unbound HCTL in plasma as a major fraction obtained by the process of extraction followed in this study. This study has revealed that although HCTL binds to plasma protein but the bound form of HCTL is not released (using this extraction solvent) and only free drug levels can be estimated. In this study, free drug levels were estimated in order to understand the less recovery obtained by the proposed method. HCTL plasma binding gave us a clue that it might be a reason for the same as free drug level was found to be very low as 11.9%. Using 1% ZnSO4 with acetonitrile is effective for clear supernatants [10] by precipitating proteins of the plasma. Considering the high plasma protein binding of HCTL, the above mixture was used as the extraction solvent in a ratio of acetonitrile and water with 1% ZnSO4 as 7:3. However, even after using this method, in this study we could not separate the protein (of plasma) bound form of HCTL. 3.1.6. Stability Studies performed to determine the drug stability in plasma indicated that HCTL was found to be unstable. The data of freeze
Table 1 Accuracy and precision of HCTL (in plasma) for the intra-day and inter-day. Theoretical concentration (nmol/ml)
0.50 2.03 4.06 8.13 32.5
Accuracy (%)
Precision (%CV)
Intra-day
Inter-day
Intra-day
Inter-day
111.54 107.48 102.17 98.94 100.03
106.61 104.80 98.69 96.79 100.13
11.30 11.88 11.66 13.37 6.98
10.95 11.88 11.17 13.56 6.94
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Table 2 Stability of HCTL in plasma at different temperatures. Nominal concentration (nmol/ml)
Freeze thaw (mean ± SD)
Short terma (mean ± SD)
Long termb (mean ± SD)
Long termc (mean ± SD)
0.50 4.06 32.5
0.5 ± 0.00 4.06 ± 0.00 32.51 ± 0.01
0.5 ± 0.28 4.06 ± 0.56 32.52 ± 2.75
0.13 ± 0.02 0.44 ± 0.06 6.91 ± 2.43
0.02 ± 0.00 0.04 ± 0.02 0.33 ± 0.03
a b c
After 6 h at room temperature. After 7 days at −86 ◦ C. After 30 days at −86 ◦ C.
Table 3 Stability of HCTL in water at two different concentrations (Conc 1: 8.13 nmol/ml and Conc 2: 32.5 nmol/ml) over the period of two months. Each value indicates mean ± SD (% stability) of concentration. No. of days
Kept at 4 ◦ C
Kept at −86 ◦ C
Conc 1 Day 0 Day 1 Day 2 Day 7 Day 60
8.15 7.91 8.83 8.45 2.20
± ± ± ± ±
Conc 2 0.17 (100%) 0.78 (97%) 0.25 (109%) 0.37 (104%) 0.07 (27%)
32.5 29.83 34.25 37.77 13.20
± ± ± ± ±
Conc 1 2.35 (100%) 2.01 (92%) 0.85 (105%) 0.67 (116%) 0.1 (41%)
8.15 7.92 8.98 8.00 6.94
± ± ± ± ±
Conc 2 0.17 (100%) 0.09 (97%) 0.38 (110%) 0.15 (98%) 0.11 (85%)
32.50 28.83 34.3 32.4 28.33
± ± ± ± ±
2.35 (100%) 1.29 (89%) 2.30 (106%) 1.06 (100%) 0.47 (87%)
Table 4 Stability of extracted HCTL in plasma over the period of one month. Nominal concentration (nmol/ml)
Day 0 (mean ± SD)
Long terma (mean ± SD)
Long termb (mean ± SD)
Long termc (mean ± SD)
2.03 4.06 32.5
2.32 ± 0.23 4.31 ± 0.22 32.60 ± 1.27
1.76 ± 0.09 3.67 ± 0.37 19.70 ± 0.70
2.28 ± 0.15 3.93 ± 0.28 23.40 ± 0.35
1.96 ± 0.10 3.45 ± 0.06 21.60 ± 0.95
a b c
After 7 days at −86 ◦ C. After 15 days at −86 ◦ C. After 30 days at −86 ◦ C.
thaw, short term and long term stability of the QC samples are shown in Table 2. Within a period of 7 days, approximately 81% of the drug was found to disappear and over 30 days, 98% of the drug degraded in QC samples. The stability of HCTL in water for two concentrations (8.13 and 32.5 nmol/ml) on 0th, 1st, 2nd, 7th and 60th day at 4 ◦ C and −86 ◦ C is shown in Table 3. Although HCTL in water was found to be stable up to 7 days both at 4 ◦ C and −86 ◦ C, HCTL degradation was observed on 60th day. The antioxidants, 0.1% ascorbate and 0.1% sodium metabisulphite could not control the deterioration of HCTL observed in water after prolong storage. Stability of the extracted HCTL in plasma at −86 ◦ C with concentrations of 2.05, 4.06 and 32.5 nmol/ml on 0th, 7th, 15th and 30th day is shown in Table 4. The results of HCTL (2.05, 4.06 and 32.5 nmol/ml) spiked in water, stored at −86 ◦ C and analyzed on 0th, 7th, 15th and 30th day showed that HCTL is stable in water for all the three concentrations up to 30 days. 3.2. Quantitative determination of HCTL in plasma HCTL was estimated in all human plasma samples and the concentration for each of them is shown in Table 5.
Table 5 Homocysteine thiolactone concentration in healthy human volunteers. S. no. (control sample)
Age/gender
HCTL conc. (nmol/ml)
1 2 3 4 5 6
30/M 43/F 30/M 24/M 36/M 33/F
0.19 0.20 0.16 0.17 0.25 0.11
4. Discussion Homocysteine thiolactone (HCTL) was discovered by serendipity in 1934 as a bi product of the digestion of methionine with hydroiodic acid, a procedure used for the determination of protein methionine [11]. In all cell types, HCTL is a product of homocysteine metabolism by methionyl-t-RNA synthetase (MetRS) [6]. In general homocysteine incorporation into the protein has been recognized as post translational, reflecting facile homocysteinylation of protein lysine residues by HCTL. The homocysteinylation leads to protein damage and has been implicated in neurodegenerative disorders, cardiovascular diseases, diabetic complications, etc. [5]. Therefore, understanding the levels of HCTL in plasma and other body fluids has been considered as an essential aspect to understand their further involvement in various disease conditions. In human serum, about half of the exogenous HCTL incorporated into protein has been reported to be released as free homocysteine after reduction with DTT [12]. As homocysteine thiolactone has been shown to react with free amino groups in proteins to form isopeptide bonds, in lysine residues [13], conventionally the total homocysteine levels were considered as an indicator by liberating the Hcy from its bound form with proteins. Considering the aliphatic nature of Hcy, the reduced Hcy was cyclized to HCTL by heating it with 6 M HCl for 30 min at 100 ◦ C. The converted HCTL was estimated by HPLC using UV at 240 nm [6]. The same research group quantified the plasma HCTL levels from its ultrafiltrate after adsorbing it in DPBS-washed charcoal, eluted by dilute acid, lyophilized and estimated using HPLC. Moreover, the results of their control experiments revealed that HCTL is stable in human plasma frozen at −86 ◦ C for at least 6 month’s time. However, its instability in human serum or plasma was explained by two reactions; enzymatic hydrolysis by homocysteine thiolactonase (paraoxonase), a calcium dependant enzyme tightly associated with HDL and
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non-enzymatic reactions with side chain amino groups of protein lysine residues [6]. In contrast to the controlled studies, we found that plasma samples spiked with HCTL were unstable even at −86 ◦ C after 7 days. We also found the disappearance of exogenously added HCTL in plasma (at the level of 32 nmol/ml) to the extent of 79% on 7th day and 99% on 30th day when the samples were kept at −86 ◦ C. Further, HCTL standards stored in water at 4 ◦ C and −86 ◦ C were compared. This comparison revealed that HCTL standards in water were stable up to 7 days at 4 ◦ C while the stability at −86 ◦ C was compromised at a period of 2 months (deteriorated beyond 10%) (Table 3). The deterioration of HCTL in water may be due to oxidation; therefore, an attempt was made with sodium metabisulphite or ascorbic acid to stabilize the same. However, in the present study we found antioxidants do not have any role in controlling the deterioration of HCTL observed in water when stored for prolonged period. In order to improve the stability of HCTL in plasma during its storage, the extraction method was slightly modified and extraction was done within 6 h from the harvesting time of plasma. Further, the stability of extracted HCTL (plasma) was conducted for a period of one month and compared with HCTL stored in water at −86 ◦ C. These experiments concluded that the new method of HCTL preservation in plasma, i.e., immediate extraction and storage at −86 ◦ C, is capable of preventing its disappearance in plasma. In this study, we used the MRM transition of 118 → 56 for the quantification of HCTL. However, other transitions such as 118 → 90 and 118 → 72 can also be combined to further improve its sensitivity in the MRM quantization. The fragmentation pattern of precursor ion leading to the formation of product ion with mass of 56 is shown in Fig. 1. It is a self charged quaternary ammonium fragment of HCTL after ring opening reaction occurred in the collision cell. The signal produced by the self-charged quaternary ammonium compounds is justifiable in the ESI-MS/MS experiments as reported in literature [14]. Enhanced HCTL synthesis is reported in human breast cancer, cystathionine synthase deficient cells or folate deficient cells [3,15,16]. Due to its ability to damage the proteins [17], HCTL has been expected to play a role in cardiovascular diseases also [3,15,18]. In our previous studies [4], we have used C 18 column (Chromolith Speed rod, Merck, Germany) for the separation of HCTL. Although the method was good enough to quantify HCTL from human vitreous humor (having trace amount of proteins), further improvement was felt to be important so as to understand its interaction with major amount of plasma proteins to determine the plasma HCTL concentrations. Due to lack of the desired sensitivity, the method was later modified to utilize hydrophilic HILIC technology for the elution of HCTL and validated. HILIC separation showed high sensitivity in the optimized analytical method. In this study, the blank plasma showed a small peak
of HCTL which is present endogenously and was quantified as 0.062 nmol/ml. Interestingly, this study has also observed a well resolved endogenous peak suspected to be d-HCTL while using HILIC separation. This peak was unresolved while reverse phase column (Chromolith Speed rod) was used. Moreover, this peak was undetectable in the standards and was only observed in spiked blood samples. Although the presence of d-amino acids are reported along with aging process [19], the relevance of our finding on the presence of d-HCTL in the plasma is yet to be ascertained. 5. Conclusion A rapid and sensitive LC–MS/MS method has been developed for the quantification of HCTL in human plasma. The method has a short run time and easy sample preparation. Moreover, this study found that collected plasma samples, if extracted immediately and stored at −86 ◦ C remain stable up to a period of one month. The assay has been successfully applied to estimate the HCTL levels in healthy human volunteers. Acknowledgement The authors would like to thank DST-FIST for providing High Precision Bio-analytical Facility (HPBAF) for this analysis. References [1] R. Clarke, L. Daly, K. Robinson, E. Naughten, S. Cahalane, B. Fowler, I. Graham, N. Engl. J. Med. 324 (1991) 1149–1155. [2] W. Gu, J. Lu, G. Yang, J. Dou, Y. Mu, J. Meng, C. Pan, Adv. Ther. 25 (2008) 914–924. [3] H. Jakubowski, L. Zhang, A. Bardeguez, A. Aviv, Circ. Res. 87 (2000) 45–51. [4] S. Barathi, N. Angayarkanni, A. Pasupathi, S.K. Natarajan, R. Pukraj, M. Dhupper, T. Velpandian, C. Muralidharan, M. Sivashanmugham, Diabetes Care 33 (2010) 2031–2037. [5] G. Chwatko, H. Jakubowski, Clin. Chem. 51 (2005) 408–415. [6] H. Jakubowski, Anal. Biochem. 308 (2002) 112–119. [7] P. Daneshvar, M. Yazdanpanah, C. Cuthbert, D.E. Cole, Rapid Commun. Mass Spectrom. 17 (2003) 358–362. [8] FDA Guidance for Industry, Bioanalytical Method Validation, 2001. [9] J. Nirmal, T. Velpandian, S.B. Singh, N.R. Biswas, V. Thavaraj, R. Azad, S. Ghose, J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 879 (2011) 585–590. [10] S. Lam, L. Boselli, Biomed. Chromatogr. 1 (1986) 177–179. [11] H.D. Baernstein, J. Biol. Chem. 106 (1934) 451–456. [12] H. Jakubowski, J. Physiol. Pharmacol. 59 (Suppl. 9) (2008) 155–167. [13] J. Perla-Kajan, T. Twardowski, H. Jakubowski, Amino Acids 32 (2007) 561–572. [14] T. Velpandian, J. Nirmal, B. Arora, A.K. Ravi, A. Kotnala, Anal. Lett. 45 (2012) 2367–2376. [15] H. Jakubowski, J. Biol. Chem. 272 (1997) 1935–1942. [16] H. Jakubowski, in: R. Carmel, D.W. Jacobsen (Eds.), Homocysteine in Health and Disease, Cambridge University Press, UK, 2001, pp. 21–31. [17] H. Jakubowski, FASEB J. 13 (1999) 2277–2283. [18] H. Jakubowski, Biomed. Pharmacother. 55 (2001) 443–447. [19] Y. Nagata, T. Akino, K. Ohno, Y. Kataoka, T. Ueda, T. Sakurai, K. Shiroshita, T. Yasuda, Clin. Sci. (Lond.) 73 (1987) 105–108.
