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WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Hammami et al.

World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 2.786

Volume 3, Issue 10, 125-135.

Research Article

ISSN 2278 – 4357

RAPID DETERMINATION OF METHOTREXATE LEVEL IN HUMAN PLASMA BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Ahmed Yusuf, Syed N. Alvi, and Muhammad M. Hammami* Clinical Studies and Empirical Ethics Department, King Faisal Specialist Hospital & Research Center, MBC-03, P.O. Box 3354, Riyadh 11211, Kingdom of Saudi Arabia.

Article Received on 11 August 2014, Revised on 29August 2014, Accepted on 20 September 2014

ABSTRACT A simple, precise, and rapid reversed-phase high performance liquid chromatography (HPLC) method for the determination of methotrexate (MTX) level in human plasma using sulfamethoxazole as an internal standard (IS) was developed and validated. Plasma samples containing

*Correspondence for Author Dr.Muhammad M.

MTX were spiked with the IS, vortex-mixed with methanol, and centrifuged. After evaporation, supernatant residue was dissolved in

Hammami

0.01 M HCl and injected into the HPLC system. The compounds of

Clinical Studies and

interest were efficiently separated on Symmetry C-8 column at room

Empirical Ethics

temperature, and detected with a photodiode array detector wavelength

Department, King Faisal

at 303 nm. The mobile phase consisted of 0.03 M dibasic potassium

Specialist Hospital & Research Center, MBC-03, P.O. Box 3354, Riyadh

phosphate (pH adjusted to 6.5 with phosphoric acid), methanol, and acetonitrile (82:14:4, v:v:v), and was delivered at a flow rate of 1.0

11211, Kingdom of Saudi

ml/min. No interference in blank plasma or of commonly used drugs

Arabia

was observed, and the detection limit of MTX was 0.02 µg/ml. The relationship between MTX concentration in plasma and peak area ratio of MTX /IS was linear (R2 ≥ 0.9997) in the range of 0.05 – 20 µg/ml. Intra- and inter-day coefficient of variations (CV) were ≤ 8.2%. Extraction recoveries of MTX and the IS were ≥ 93%, whereas Intra-day and inter-day bias (relative recovery) of MTX measurement was 8.0% to 0.8% using quality control samples and -4.0% to 1.2% as determined by back calculation from peak area ratios of the calibration curves. The method was used to assess the stability of MTX in human plasma under conditions generally encountered in clinical laboratory. Further, it was successfully applied to measure MTX level in samples obtained from a patient on MTX therapy.

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KEY WORDS: Methotrexate (MTX), Sulfamethoxazole, Human plasma, HPLC. INTRODUCTION Methotrexate (MTX, 2, 4-diamino- N10-methyl folic acid) is an anticancer drug widely used in treatment of acute lymphoblastic leukemia in adults and children.

[1-2]

MTX is generally

well absorbed within 1-2 hours after ingestion. Its mean oral bioavailability is about 60% at doses ≤30 mg/m2, but significantly lower at doses ≥80 mg/m2.Inleukemic pediatric patients, oral absorption is dose dependent and bioavailability varies widely.[3-4] In order to monitor MTX level in patients, various analytical methods have been developed, including those based on enzyme inhibition and competitive protein binding, enzyme multiplied immunoassay, radioimmunoassay, and microbiological, fluorimeter or high performance liquid chromatography (HPLC) assays.

[5-11]

HPLC is one of the most reliable

and predominantly used technique for the measurement of MTX level in human plasma.[1216]

The majority of the reported HPLC assays used relatively large plasma volumes (up to 1

ml) and/or multi-step, liquid or solid-phase extraction procedures. Recently, a micro flow liquid chromatography coupled with mass spectrometry (LC-MS/MS) was reported.

[17]

However, this method may not be feasible for low budget laboratories. Further, its applicability for patient sample analysis has not been documented. In the present study, we describe a simple, precise, and rapid HPLC assay that requires 0.2 ml human plasma, and is based on simple protein precipitation. The method was fully validated and used to determine methotrexate stability and methotrexate level in plasma samples obtained from patients on methotrexate therapy. MATERIAL AND METHODS Apparatus: Chromatography was performed on a Waters Alliance HPLC 2695 (Waters Associates Inc., Milford, MA, USA) consisting of a quaternary pump, autosampler, column thermostat, and photodiode array detector. A reversed-phase column Symmetry C-8 (4.5 x 150 mm, 5-μm) and a guard pak pre-column module with a Nova-pak C18 4-μm insert were used for the separation. Data were collected with a Pentium IV computer using Empower Chromatography Software. Chemical and reagents: All reagents were of analytical-reagent grade unless stated otherwise. Methotrexate (USP reference standard) was purchased from US Pharmacopeia,

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Rockville, Maryland, USA. Sulfamethoxazole and dibasic potassium phosphate were purchased from Sigma-Aldrich Co., St. Louis, MO, USA. Acetonitrile and methanol (both HPLC grade), phosphoric acid, and sodium hydroxide were purchased from Fisher Scientific, Fairlawn, NJ, USA. Hydrochloric acid was purchased from E-Merck, Darmstadt, Germany. HPLC grade water was prepared by reverse osmosis and was further purified by passing through a Synergy Water Purification System (Millipore, Bedford, MA, USA).Drug-free human plasma was obtained from the blood bank of King Faisal Specialist Hospital & Research Centre (KFSHRC) Riyadh, Saudi Arabia. Chromatographic conditions: The mobile phase composed of 0.03 M dibasic potassium phosphate (pH adjusted to 6.5with phosphoric acid), methanol and acetonitrile (82:14:4, v:v:v). Before delivering into the system, the mobile phase was filtered through 0.45 μm polyetersulfone membrane and sonicated under vacuum for 5 minutes. The analysis was carried out under isocratic conditions using a flow rate of 1.0 ml/min at ambient temperature and a run time of 10 minutes. A photodiode array detector set at 303 nm was used. Preparation of standard and quality control samples: Stock solutions (1mg/ml) of MTX in 0.01 N sodium hydroxide and sulfamethoxazole (internal standard, IS) in methanol were prepared. They were diluted with blank human plasma and mobile phase, respectively, to produce working solutions of 100 μg/ml and 500 μg/ml, respectively. Nine calibration standards in the range of 0.05 – 20 μg/ml and four quality control (QC) samples (0.05, 0.15, 8, and 18 μg/ml) were prepared in human plasma. Calibration standards and QC samples were vortexed for one minute and 0.2 ml aliquots were transferred into micro centrifuge tubes and stored at -20 ºC until used. Sample preparation: Aliquots of 0.2 ml of calibration standards, QC samples, or patient’s samples were allowed to equilibrate to room temperature. To each tube, 70 μl of the IS working solution was added and the mixture was vortexed for 10 seconds. After the addition of 0.8 ml of methanol, the samples were again vortexed for one minute and then centrifuged for 10 min at 13600 rpm. 0.8 ml of the clear supernatant was transferred into a 5-ml glass culture tube and evaporated to dryness under a gentle nitrogen stream at room temperature. The dry residue was dissolved in 300 µl of 0.01 M HCl and 80 μl were injected into the HPLC system.

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Stability studies: Three QC samples in concentration 0.05, 0.15, and 18μg/ml were used for MTX stability studies. Five aliquots of each QC sample were extracted and immediately analyzed (baseline), Five aliquots were allowed to stand on the bench-top for 24 hours at room temperature (counter stability, 24 hours at room temperature) and five aliquots were stored at -20 ºC for six weeks (long term freezer storage stability) before processing; and five aliquots were processed and stored for 24 hours at room temperature or 48 hours at -20 ºC before analysis (autosampler stability). Fifteen aliquots of each QC sample were stored at -20 ºC for 24 hours. They were then left to completely thaw unassisted at room temperature. Five aliquots of each sample were extracted and analyzed and the rest returned to -20 ºC for another 24 hours. The cycle was repeated three times (freeze-thaw stability). Method validation The method was validated according to standard procedures described in the US Food and Drug Administration (FDA) bioanalytical method validation guidance.

[18]

The validation

parameter included: specificity, linearity, accuracy, precision, recovery and stability. RESULTS AND DISCUSSION Optimization of chromatographic conditions Figure 1 depicts the chemical structures of MTX and the IS sulfamethoxazole. The optimal experimental conditions consisted of a mobile phase composed of 0.03 M dibasic potassium phosphate (pH = 6.5 ± 0.02), methanol, and acetonitrile (82:14:4, v:v:v) and a flow rate of 1.0 ml/min. Under these conditions MTX, sulfamethoxazole, and unknown components of plasma exhibited a well-defined separation within 10 minutes run. The retention times of MTX and sulfamethoxazole were around 5.9 and 7.8 minutes, respectively. Specificity In order to confirm specificity of the assay, we screened six batches of blank plasma and eight frequently used medications (ranitidine, acetaminophen, ibuprofen, nicotinic acid, ascorbic acid, caffeine, diclofenac, and omeprazole) for potential interference. No interference was found in plasma and none of the drugs co-eluted with MTX or the IS. Figure 2 depicts a representative chromatogram of drug free human plasma used in preparation of standard and QC samples.

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Table 1: Back calculated methotrexate concentrations from ten calibration curves Calculated Level(μg/ml) Nominal Level CV (%) Bias (%) (μg/ml) Mean SD 0.05 0.049 0.004 8.2 -2.0 0.1 0.096 0.007 7.3 -4.0 0.2 0.193 0.016 8.3 -3.5 0.5 0.483 0.019 3.9 -3.4 1.0 0.985 0.029 2.9 -1.5 2.5 2.530 0.081 3.2 1.2 5.0 5.052 0.108 2.1 1.0 10.0 9.929 0.139 1.4 -0.7 20.0 20.016 0.049 0.2 0.1 SD, standard deviation. CV, standard deviation divided by mean measured concentration x100. Bias = (mean measured concentration – nominal concentration divided by nominal concentration) ×100. Table 2: Intra-day and inter-day precision and bias of methotrexate assay Intra-day (n = 10)

Inter-day (n=20)

Mean Mean Measured CV Bias Measured CV Bias SD SD Level (%) (%) Level (%) (%) (µg/ml) (µg/ml) 0.05 0.046 0.003 6.5 -8.0 0.049 0.004 8.2 -2.0 0.15 0.140 0.007 5.0 -6.7 0.139 0.006 4.3 -7.3 8 7.763 0.129 1.7 -3.0 7.917 0.188 2.4 -1.0 18 17.567 0.358 2.0 -2.4 18.148 0.681 3.8 0.8 SD, standard deviation. CV, standard deviation divided by mean measured concentration x100. Bias = (mean measured concentration – nominal concentration divided by nominal concentration) × 100. Nominal Level (µg/ml)

Table 3: Recovery of methotrexate and internal standard from 0.2 ml human plasma Nominal Level (µg/ml) Methotrexate 0.05 0.15 8 18 Internal Standard 35

Human Plasma

Mobile Phase

Recovery (%)

5571 (266) 16691 (5410 963576 (42096) 2025633 (89211)

5572 (301) 16741 (242) 1030136 (3103) 2178564 (2206)

100 100 94 93

1930530 (124451)

2033278(4759)

95

Data represent mean peak area (SD), n= 5. Recovery is ratio of mean peak area in human plasma divided by mean peak area in mobile x 100

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Table 4: Stability of methotrexate in processed and unprocessed human plasma Storage condition None (Baseline)

Processed Samples 24 hr, RT

Nominal Level (µg/ml) 0.05 0.15 18

Measured level (µg/ml) 0.050 (0.002) 0.162 (0.003) 18.925 (0.105)

0.05

0.049 (0.002)

Stability (%)

98

0.15 0.149 (0.005) 92 18 19.061 (0.088) 101 48 hr, -20 ºC 0.05 0.051 (0.004) 101 0.15 0.154 (0.007) 95 18 18.398 (0.061) 97 Unprocessed Samples 0.05 0.051 (0.003) 102 24 hr, RT 0.15 0.150 (0.002) 93 18 18.082 (0.415) 101 6 wks, -20 ºC 0.05 0.044 (0.004) 88 0.15 0.128 (0.005) 79 18 17.746 (0.051) 94 Unprocessed Samples 0.05 0.045 (0.004) 90 FT Cycle-1 0.15 0.144 (0.008) 89 (-20 ºC, RT) 18 18.813 (0.083) 99 0.05 0.050 (0.002) 100 FT Cycle-2 0.15 0.147 (0.003) 91 (-20 ºC, RT) 18 19.017 (0.076) 100 0.05 0.044 (0.002) 89 FT Cycle-3 0.15 0.140 (0.003) 97 (-20 ºC, RT) 18 19.044 (0.092) 101 Mean peak area (SD), n=5. Stability (%) =Mean measured level at the indicated storage condition divided by mean measured level at baseline x 100. Data report mean peak area (SD), n= 5. RT, Room Temperature (24 ºC). FT, Freeze-thaw.

Fig. 1 Chemical structures of methotrexate and sulfamethoxazole (IS).

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Fig. 2 Representative chromatogram of a drug-free human plasma. The arrows indicate the retention times of methotrexate (5.9 min) and sulfamethoxazole (Internal standard, IS) (7.8 min).

Fig. 3 Overlay of chromatograms of extracts of 0.2 ml human plasma spiked with the internal standard (IS) and one of ten concentrations of methotrexate, 0.0, 0.05, 0.1, 0.2, 0.5, 1.0, 2.5, 5.0, 10.0, and 20.0 μg/ml.

Fig. 4A typical chromatogram of a sample obtained from a patient receiving methotrexate therapy.

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Limit of Detection & Quantification and Linearity: The limit of quantification (LOQ) was defined as the lowest concentration on the calibration curve that can be determined with acceptable precision and accuracy (i.e., coefficient of variation and bias ≤ 20%). The LOQ of MTX in human plasma was 0.05 μg/ml. The limit of detection (LOD, ≥ 3 S/N) was 0.02 μg/ml. Linearity of MTX was evaluated by analyzing ten curves of nine calibration standards over the range (0.05-20 μg/ml) prepared in human plasma. Calibration curves were linear with an R2≥ 0.9997. Figure 3 shows an overlay of chromatograms of a typical calibration curve.The peak area ratios were subjected to regression analysis. The mean regression equation was Y = 0.9997 X – 0.0088. The suitability of the calibration curves was confirmed by back-calculating the concentration of MTX in human plasma from the calibration curves (Table 1). All calculated concentrations were well within the acceptable limits. Precision and bias (inaccuracy) The intra-day and inter-day precision and bias of the method were evaluated by analyzing four different QC samples (0.05, 0.15, 8, and 18 μg/ml). The intra-day precision and bias (n = 10) ranged from 1.7% to 6.5% and from -8.0% to-2.4%, respectively. The inter-day precision and bias were determined over three different days (n = 20) and ranged from 2.4% to 8.2% and from -7.3% to 0.8%, respectively. The results are summarized in Table 2. Recovery The absolute recovery of MTX was assessed by direct comparison of peak areas from plasma and mobile phase samples, using five replicates for each of four QC samples (0.05, 0.15, 8, and 18 μg/ml). Similarly, the recovery of the IS was determined by comparing the peak areas of the IS in 5 aliquots of human plasma spiked with 100 μl of IS (35 μg/ml) with the peak areas of equivalent samples prepared in mobile phase. The results are presented in Table 3. Recovery was ≥ 93% for MTX and 95% for the IS. Robustness The robustness of a method is a measure of its capacity to remain unaffected by small variations in chromatographic conditions. It gives an indication of the reliability of the method during routine applications. The robustness of the current assay was evaluated by altering the strength of potassium phosphate buffer (±0.01 M), pH(±0.02%), and the proportion of methanol (±2%) in the mobile phase. No significant changes in peak area, peak shape, or retention time were observed; indicating the robustness of the method.

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Stability: Stability of analyte in biological matrices is an important pre-analytical variable. It is necessary to perform stability studies of the analyte and IS to determine the range of appropriate conditions and time of storage. Methotrexate and IS stability in processed and unprocessed plasma samples was investigated. Processed samples containing MTX (0.05, 0.15, and 18 μg/ml) were stable for at least 24 hours at room temperature (≥ 92%) and 48 hours at -20°C (≥ 95%).Methotrexate in unprocessed plasma samples was stable for at least six weeks at -20°C (≥ 79%), and after three freeze-and thaw cycles (≥ 89 %). (Table 4). Similarly, MTX (1 mg/ml in 0.01 N sodium hydroxide) and IS stock solutions (1mg/ml in methanol) were stable for at least 48 hours at room temperature (≥ 100%) and at least seven weeks at -20°C ( ≥ 90%).Further, no significant change in chromatographic behavior of MTX or the IS was observed. Application to a patient sample Figure 4 depicts a typical chromatogram of sample collected from a patient with acute lymphoblastic leukemia on methotrexate therapy. The sample was collected on the 44th day of treatment. The measured level of methotrexate was11.5μg/ml. CONCLUSION The described HPLC assay is precise and rapid. It requires only 0.2 ml plasma and utilizes a simple and convenient method for sample preparation. The assay was applied to monitor stability of MTX under various conditions encountered in the clinical laboratories. Further, it was successfully used to determine MTX in plasma samples obtained from a patient on MTX therapy. REFERENCES 1. Widemann BC, Adamson PC, Understanding and managing methotrexate nephrotoxicity. The Oncologist. 2006; 11: 694-703. 2. Panetta JC, WallA, Pui

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