INTRODUCTION:

Cycloserine (fig. 1) is a broad spectrum antibiotic that is produced by a strain of Streptomyces orchidoceous and has also been synthesized. Its main application is the treatment of active pulmonary and extra pulmonary tuberculosis¹. The chemical name of cycloserine is D-4-Amino-3-isoxazolididone². Cycloserine is an analog of the amino acid ‘D-alanine’. It interferes with an early step in bacterial cell wall synthesis in the cytoplasm. Very few methods of analysis for cycloserine are reported in literature and most of them are HPLC methods^{1, 3-5}. Some methods required complex procedure of derivatization for estimation^{1, 3}. To the best of our knowledge there is only one LC-MS/MS method reported in literature for estimation of cycloserine⁶. LC-MS/MS methods have advantages over HPLC-UV methods in terms of are fast and more specific technique of drug analysis. Our aim was to develop an accurate, specific and sensitive method of analysis for the estimation of cycloserine from human plasma using LC-MS/MS. The method was developed using Acyclovir as an internal standard (fig. 2) and validated.

Cycloserine is water soluble drug, and hence extraction method was developed using solid phase extraction technique to ensure good sample clean up. The method was validated by evaluating the precision, accuracy and for other validation parameters as mentioned in regulatory guidance⁷.

Fig. 1 Structure of Cycloserine

Fig. 2 Structure of Acyclovir

MATERIALS AND METHODS:

Apparatus and Chromatographic conditions:

The system used was Shimadzu LC-VP HPLC System consisting of LC-10AD Prominence pump, SIL-HTc autosampler, CTO 10ASvp column oven and DGU-14A degasser was used to setting the reverse-phase LC conditions. The separation of cycloserine and internal standard was performed on Inertsil ODS-3V, 4.6 X 250 mm, 5µ column at 40°C. The mobile phase consisted of Milli-Q water : Acetonitrile : formic acid (30:70:0.3 v/v). Flow rate of mobile phase was kept at 1ml/min. with the total chromatographic run time of 2.6 minutes. The auto-sampler temperature was maintained at 4°C. Injection volume was 5µl and rinsing solution was water and acetonitrile in the volume ratio of 50:50 . Ionization and detection of cycloserine and IS was accomplished on triple quadrupole mass spectrometer, MDS SCIEX API 4000 (Toronto Canada) equipped with electrospay ionisation operating in positive ion mode. Quantitation was performed using MRM mode to monitor protonated Precursor → product ion(m/z) transitions for cycloserine 103.3→75.200 and for Acyclovir 226.150→152.100. The optimised source dependent parameters were nebuliser gas- 50 psig, heater gas- 40 psig, ion spray voltage- 4500V, Turbo heater temperature-600°C, interface heater-on, Curtain gas was nitrogen 20 psig and CAD- 5 psig. The compound dependent parameters like declustering potential, entrance potential, collision energy and cell exit potential were 49, 5, 11,13 V respectively for cycloserine and 56, 8, 40, 10V respectively for IS. Data processing and chromatographic integration was carried out by using ‘Analyst software version 1.5.1’. The nitrogen evaporator used to evaporate the samples was procured from Takahe Analytical Instruments. Deep freezers used for storage of plasma samples were procured from SANYO (JAPAN) were used.

Chemicals and Reagents:

Cycloserine and acyclovir working standards were obtained from Macleods Pharmaceuticals Ltd, Mumbai, India. Water was deionized and further purified with Milli-Q system (Millipore USA), Methanol (HPLC grade) was supplied by J. T. Baker (U.S.A.), Acetonitrile (HPLC grade), formic acid (AR grade) and ammonia (HPLC grade) was supplied by Thomas Baker (INDIA). SPE cartridges used for sample preparation were Water Oasis MCX cartridges 1CC /30 mg.

Fresh frozen plasma containing dipotassium EDTA as an anticoagulant was used during validation and study sample analysis was collected in-house in Macleods Pharmaceuticals Ltd, Mumbai India. Plasma was stored at -20°C before sample preparation and analysis.

Standards and Working Solutions:

Stock standard solutions of Cycloserine containing 3000 µg/ml was prepared by dissolving pure standard in 50 % methanol and Internal standard 1000 µg/ml was prepared by dissolving 10 mg of pure standard in 1 ml formic acid further 1 ml of water was added to it and then volume was made up to 10 ml with methanol. Intermediate dilutions and IS spiking dilutions were prepared from respective stock solutions by dilution with 50% methanol in water. Calibration standards were prepared in the range of 0.2 µg/ml to 16.0 µg/ml of cycloserine at eight concentration levels. Quality control standards were prepared at three different levels low (0.559 µg/ml), medium (8.227 µg/ml) and high (13.712 µg/ml).

Sample Treatment:

50 µl of IS solution was added to 100µl of plasma sample and vortexed. Then 1.9 ml of 2 % formic acid in water was added to it and vortexed. These plasma samples were extracted on OASIS MCX SPE cartridges. Conditioned the OASIS MCX cartridges with 1 ml methanol followed by 1 ml 2% formic acid in water. Then the plasma samples were loaded on the cartridges. The cartridges were washed twice with 1 ml of water, twice with 1 ml of 2 % formic acid in methanol followed by twice 1 ml of methanol. The samples were eluted twice with 1 ml of 1% ammonia in methanol. The samples were evaporated to dryness at 50°C under nitrogen and reconstituted with 2 ml of 70 % acetonitrile in water.

RESULTS AND DISCUSSIONS:

Optimization of Chromatographic Conditions and Sample Clean-up:

The successful analysis of the analyte in biological fluids using LC-MS/MS method relies on the optimization of sample preparation, chromatographic separation and post column detection etc. The method development was initiated to achieve adequate selectivity, sensitivity and minimize overall analysis time and using small plasma volume for processing. Optimum mass acquisition parameters were obtained by direct infusion of 500 ng/ml solution for cycloserine at flow rate of 10µl/min. This was done by maintaining optimized declustering potential and ion spray voltage at 49 V and 4500 V respectively. The present study was conducted using positive mode of ESI as the analyte can be easily protonated. The ESI Q1 mass spectrum of cycloserine and IS was dominant with protonated precursor (M+H)⁺ions as the drug is protonated in acidic pH. The most stable and consistent product ion for cycloserine was observed at m/z 75. For Acyclovir the protonated guanine species (m/z 152.1) was the most abundant ion seen in the product ion mass spectra. All the state file parameters were optimized to obtain a consistent and adequate response for the analyte and IS. Chromatographic conditions of the analyte and IS was initiated under isocratic conditions to obtain adequate response, sharp peak shape and a short run time. It was observed that the pH of the buffer was crucial for protonation of cycloserine. Low pH is necessary for the protonation of amino group of cycloserine⁴. Various mobile phase containing combination of volatile acids and buffers like formic acid, acetic acid, ammonium acetate with acetonitrile and methanol were tried to get good, stable and reproducible response. Finally water: acetonitrile: formic acid combination in proportion of 30: 70: 0.3 v/v was selected. Various columns, BDS C8, 250 × 4.6, 5µ , Hypurity Advance 50 × 4.6, 3µ , Peerless Basic C8 150 × 4.6, 5µ, Inertsil ODS 150 × 4.6, 5µ, Inertsil ODS-3V 250 × 4.6, 5µ, Zorbax phenyl 250 × 4.6, 5µ were evaluated for suitable peak shape, response and retention of the analyte and IS. Best results were observed with Inertsil ODS-3V 250 × 4.6, 5µ. The SPE technique was optimized to obtain clean samples. Different techniques like protein precipitation, liquid-liquid extraction and solid phase extraction were used for sample clean-up. Since cycloserine is highly water soluble, recovery in liquid –liquid extraction was very low. Sample preparation using de-proteination resulted in more matrix effect. The extraction procedure was finally optimized using solid phase extraction MCX cartridges 1cc / 30 mg. During sample preparation washing step was optimized using with 1 ml water, 1 ml 2 % formic acid in methanol and 1 ml methanol twice to get cleaner samples so as to reduce matrix effect. Elution of the sample was carried out twice with 1ml of 1 % ammonia in methanol to get maximum recovery.

Acyclovir was used as an internal standard in the present study as it showed similar chromatographic behavior and both the drugs were quantitatively extracted via solid phase extraction.

Method Validation:

Carry over effect of the autosampler was evaluated by sequentially injecting solutions of mobile phase and blank after extracted high concentration sample containing cycloserine and internal standard (concentration equivalent to 1.68 times of ULOQ) and its aqueous recovery comparison sample. No significant carry over was observed when rinsing cycle before and after with 500µl of rinsing solution was applied.

Fig. 3 Representative chromatogram of blank sample

Specificity:

For specificity and selectivity testing, ten different lots of plain plasma and six lots of hemolysed plasma were analysed to ensure that no endogenous interference at the retention time of Cycloserine and IS. LLOQ level samples from respective plasma lots were prepared and analysed with plasma blanks. In all plasma blanks, the area response at the retention time of cycloserine was less than 20 % of LLOQ response and at the retention time of IS, the area response was less than 5 % of the mean IS response in LLOQ. Representative chromatogram of blank sample is given in fig. 3.

Sensitivity:

For Lower limit of quantification (LLOQ), one set of six LLOQ samples prepared in plain plasma and one set of six LLOQ samples prepared in hemolysed plasma along with one set of calibration curve in plain plasma. The lowest limit of quantification was set at the concentration of the 0.200 µg/ml from plasma (fig 4.). The precision and accuracy at LLOQ was found to be 5.08% and 105.60 % for plain plasma and 15.52 % and 98.50 % for plasma obtained from hemolysed blood.

Fig. 4 Representative chromatogram of LLOQ sample

Matrix Effect:

Matrix effect may arise due to co-elution of some unintended components present in biological samples or which are added as part of analysis. These components result in ion suppression / enhancement, decrease / increase in sensitivity of analytes over a period of time, increase in baseline, imprecision of data, drift in retention time and distortion or tailing of chromatographic peak. Thus assessment of matrix effect constitutes an important and integral part of validation for quantitative LC-MS/MS method. The % CV of IS normalized matrix factor at LQC, MQC and HQC was 6.18 %, 6.02 % and 5.28 % respectively.

Linearity:

The linearity of the method was determined by analysis of eight point calibration standards. Three linearity curves were analyzed. A regression equation with a weighting factor of1/x and 1/x² of ratio of drug to internal standard concentration were checked for better results in terms of accuracy. Finally 1/x²was used to produce the best fit for the concentration-detector response relationship. Correlation coefficients (r²) were greater than 0.98 in the concentration range of 0.200 µg/ml to 16.000µg/ml (ULOQ fig. 5). Accuracy of all calibration standards was within 85-115 % except LLOQ where it was 80-120 %.

Fig. 5 Representative chromatogram of highest concentration sample

Precision and Accuracy:

The precision of the assay was measured as the percent coefficient of variation over the concentration range of LLOQ QC, LQC, MQC and HQC samples during the course of validation. The accuracy of the assay was defined as the absolute value of the ratio of the calculated mean values of LLOQ QC, LQC, MQC and HQC samples to their respective nominal values, expressed in percentage. Six replicates of each QC sample were analyzed together with a set of calibration standard. For determining the intra-day accuracy and precision, replicate analysis of plasma samples was performed on the same day. The inter-day accuracy and precision were assessed by analysis of three precision and accuracy batches on two different days.

The obtained precision and accuracy (inter and intra-day) are presented in Table 1. The result showed that the method is accurate as the inter-day accuracy ranged from 90.70 % to 100.90 %and the precision around the mean value ranged from 5.82 % to 8.54 %. Intra-day accuracy ranged from 90.10 % to 97.36 % and precision ranged from 3.24 % to 9.21 %.

Table 1: Inter and Intra-Day Accuracy and Precision of Cycloserine

QC levels	Mean Accuracy (%)	Precision (%RSD)
Intra day (n=12)
LLOQ QC	90.10	9.21
LQC	90.41	7.50
MQC	97.36	3.24
HQC	97.24	4.28
Inter day (n=18)
LLOQ QC	90.70	8.06
LQC	93.31	8.54
MQC	100.88	5.82
HQC	100.90	6.33

Recovery:

The recovery study was performed by comparing processed QC samples of three different concentrations with aqueous recovery comparison samples representing 100% extraction. The recovery at low, medium and high quality control level was found to be 63.69 %, 72.84 % and 73.08 % respectively. Recovery of IS was 83.52 %.

Hemolysis Effect:

To determine the effect of hemolysis, ten hemolysed plasma blanks along with LLOQ samples and QC samples at three concentrations (Low, Medium and High Concentration) were prepared. Six replicates of each QC sample were analyzed together with a set of calibration curve prepared in plain plasma. None of the blank sample showed any significant interference at the retention time of analyte and IS. The accuracy ranged from 89.16 % to 90.58 % and precision ranged from 4.72 % to 9.22 %.

Dilution Integrity:

Dilution integrity quality control samples were prepared by spiking approximately 1.68 times (26.828 µg/ml) the highest standard concentration of Cycloserine (16 µg/ml). Six samples of dilution integrity samples were processed by diluting them twice and another six samples by diluting them four times with plain human plasma. These quality control samples were analysed along with the calibration curve standards and the quality control sample concentrations were calculated using appropriate dilution factor. The method was precise and accurate for both the dilution factor.

Stability Studies:

The stability of cycloserine and IS was investigated in the stock and working solutions, in plasma during storage, during processing, after three freeze-thaw cycles and in the final extract. The stability samples were compared with freshly prepared calibration curve and quality control samples. All stability exercises were carried out on six sets of LQC and HQC samples except for Long term stability where six sets of LQC, MQC and HQC samples were used for stability testing. Analyte and IS were considered stable when the change of concentration was ± 15 % of nominal value.

Freeze Thaw Stability:

For freeze thaw stability, retrieval of samples stored at -50°C was carried out after 24 hours for first FT cycle and then five more FT cycles were carried out after at least 12 hours of freezing for each cycle. The samples were found to be stable even after six FT cycles. Summary of stability data is presented in Table 2.

Dry Extract Stability:

The dry extract stability at room temperature after extraction from plasma only up to stage of evaporation to dryness was performed by storing these samples at room temperature without reconstitution for 5 hours. The samples were found to be stable for five hours. Summary of stability data is presented in Table 2.

Post Preparative Stability:

The post preparative stability of extracted plasma samples was evaluated after final reconstitution step. The samples were stored at room temperature for 4 hours and results showed that samples were found to be stable for 4 hours. Summary of stability data is presented in Table 2.

Bench top stability:

Bench Top stability was evaluated using six sets each of LQC and HQC by placing quality control samples at bench top for 7 hours. The plasma samples were found to be stable for 7 hours at room temperature. Summary of stability data is presented in Table 2.

Autosampler Stability:

In assessing the auto sampler stability, QC samples placed in the autosampler, were injected after 31 hours. The samples were found to be stable for 31 hours at 4°C. Summary of stability data is presented in Table 2.

Long Term Stability in Plasma:

For long-term stability, concentrations obtained are compared with the results of 1^st day of analysis of bulk-spiked samples. QC samples at low, medium and high concentration were processed after 175 days of storage in deep freezer maintained at below -50°C and checked for the stability. The samples were found to be stable for 175 days. Summary of this stability data is presented in Table 2.

Stock Solution Stability:

Bench top stock solutions stability and refrigerator stock solutions stability were evaluated by injecting six replicates of stock dilutions of both stability and comparison stock solution of Cycloserine and Acyclovir. The stock solutions of Cycloserine and Acyclovir were found to be stable for 49 hours at room temperature and in refrigerator stock solutions were found to be stable for 12 days 19 hours.

Bioequivalence Study:

The validated method was further applied to study sample analysis. Design of the study was an open label, single dose, crossover bioequivalence study. The study was conducted as per the Independent Ethics Committee approved study protocol. The study was carried out on 24 healthy, adult human subjects under fasting conditions. After LC-MS/MS analysis the plasma cycloserine concentrations (µg/ml) were subjected to statistical analysis. The test and reference products were found to be bioequivalent. The comparative linear plots of cycloserine mean plasma concentrations (µg/ml) for test and reference product Vs Time were given in fig. 6.

Fig.6 Comparative Linear Plot of Cycloserine Mean Plasma Concentration (µg/ml) Vs Time (hour)

CONCLUSION:

A simple sensitive, selective, precise and accurate LC-MS/MS method for the determination of cycloserine from human plasma was developed. The method was validated and can be used for the routine analysis for estimating cycloserine from human plasma.

ACKNOWLEDGEMENTS:

The authors thank Macleods Pharmaceuticals Ltd, Mumbai, for providing all the working standards, chemicals, Laboratory instruments and facility to carry out this work.

REFERENCE:

1. David V, Ionescu M, Dumitrescu V, Determination of cycloserine in human plasma by high-performance liquid chromatography with fluorescence detection, using derivatization with p-benzoquinone, Journal of Chromatography B, Biomedical Sciences and Applications. 761(1); 2001:27-33.

2. Merck Index, Merck and Co. Inc. White house Station, NJ, USA. (2006); 14^th edition :2751: pp 461

3. Kim Y. G., Lee Y. J., Shim C. K. et al Bioequivalence assessment of closerin capsule to Dura seromycin capsule of cycloserine after a single oral dose administration to healthy male volunteers, International Journal of Clinical Pharmacology and Therapeutics., 38: 2000:461-466

4. Pendela M, Dragovic S, Bockx L, Hoogmartens J, Van Schepdael A, Adams E., Development of a liquid chromatographic method for the determination of related substances and assay of d-cycloserine, Journal of Pharmaceutical and Biomedical Analysis. 47(4-5); 2008: 807-811

5. Karthikeyan K, Arularasu GT, Rmadas R, Pillai KC, Development and validation of indirect RP-HPLC method for enantiometric purity determination of D-cycloserine drug substance, Journal of Pharmaceutical and Biomedical Analysis. 54(4); 2011: 850-854.

6. Kim S., LC/MS/MS method for the simultaneous determination of cycloserine, mannitol, rifampicin as markers in Caco-2 culture model, available on, www.aaps.org/abstracts/AM_2007/ AAPS2007-001262 [Accessed on April 15, 2011].

7. Guidance for Industry, Bioanalytical Method Validation (2001) Food and Drug Administration, Centre for Drug and Research (CDER).

Received on 17.11.2011 Modified on 14.12.2011

Asian J. Research Chem. 5(1): January 2012; Page 44-49

Stability	QC level	Mean Accuracy (%)	Precision (% CV)	Stability duration
Bench Top	LQC	95.47	4.28	7 hours
Bench Top	HQC	91.35	4.19	7 hours
Freeze thaw	LQC	93.65	7.37	6 cycles
Freeze thaw	HQC	88.77	9.49	6 cycles
Dry Extract	LQC	88.86	3.06	5 hours
Dry Extract	HQC	94.62	0.82	5 hours
Post preparative	LQC	85.69	3.20	4 hours
Post preparative	HQC	90.22	5.94	4 hours
Auto sampler	LQC	91.81	5.54	31 hours
Auto sampler	HQC	99.21	4.90	31 hours
Long Term Plasma Stability	LQC	94.24	4.61	175 days
	MQC	96.12	7.04
	HQC	96.23	2.09