Determination of pyrazinamide in human by high performance liquid chromatography.SN Revankar, ND Desai, AB Vaidya, AD Bhatt, B Anjaneyulu
Medical Department, Hindustan Ciba Geigy Research Centre, Bombay, Maharashtra.
A facile and sensitive high performance liquid chromatographic (HPLC) technique has been developed for the determination pyrazinamide (PZA) in human plasma. Nicotinamide(NIA) is used as internal standard(IS). Plasma is deproteinized with 0.7 M perchloric acid; clear supernatant is neutralized with 1M NaOH and injected onto HPLC. The separation of pyrazinamide and the internal standard is carried out on a Supelco LC-18 (DB) column with a basic mobile phase. Pyrazinoic acid, the major metabolite, other anti-tuberculous drugs and endogenous components do not interfere with measurement of pyrazinamide. The limit of detection of pyrazinamide with this method is 0.2 mg/0.2 ml plasma (CV 8.2%).
Keywords: Adult, Antitubercular Agents, blood,Biological Availability, Chromatography, High Pressure Liquid, methods,standards,Human, Male, Niacinamide, blood,Pyrazinamide, blood,pharmacokinetics,Reproducibility of Results,
Pyrazinamide (PZA), the amide of pyrazinoic acid derived from pyrazine (1,4-diazine) is an important anti-tuberculosis agent. The determination of PZA levels in biological fluids was carried out earlier by spectroscopic methods, and gas chromatographic-mass spectrometric technique. A HPLC technique reported earlier had a step of very tedious extraction. Another HPLC technique also involved a preliminary extraction of the drug and an internal standard, paracetamol, from acidified rabbit plasma samples (pH 4.2).
The present article describes a method developed by us employing reversed-phase HPLC with UV detection of PZA using nicotinamide (N IA) as an internal standard (IS).
Materials: The following reagents were used: Pure PZA standard was obtained from Pharma Development Department, Hindustan CIBA-GEIGY Ltd. Bombay; nicotinamide-Sigma. KH2P04 BDH: Na2HPO4.2H20- Proanalysi. SM: perchloric acid about 60% Proanalysi, E. Merck; methonal Uvasol, E. Merck: and sodium hydroxide-GR.SM.
Glass distilled water was used. Buffer (pH 7.4) was prepared with 19.7 ml of 0.2 M KH2PO4 and 80.3 ml of 0.02 M Na2HPO4.2H120and filtered before use through a Millipore filter (pore size 0.45 urn; Waters Assoc.)
Instrumentation: Varian (Zug, Switzerland) Model 5000 ternary solvent delivery system was employed with an UV-Visible variable wave-length detector (Spectra monitor DIDC/Milton Roy, U.S.A.) set at 268 nm coupled to an electronic integrator (Shimadzu, chromatopac C-R3 A Japan)
Chromatographic conditions: Separation was carried out on a reversed phase 40-urn LC-18 5 cm Pelliguardcolumn linked to Supelco LC-18 (DB) (5 U 150 mm x 4.6 mm I.D) analytical column using 0.02 M phosphate buffer (pH 7.4)- methanol (96.8: 3.2, v/v) as a mobile phase pumped at flow-rate of 1.5 ml/min. PZA and internal standard (IS) eluted at 5.2 and 6-0 minutes respectively. Calibration and analysis were based on standard/internal standard peak area integration ratio. Pyrazinoic acid, rifampicin, isoniazid and acetylisoniazid were also chromatographed with the above conditions.
Analytical procedure: Standard solutions prepared as follows: Stock solutions of PZA and NIA were prepared by dissolving 5 mg of each compound separately in 50 ml glass distilled water to yield a solution of 100 ?g/ml concentration. The stock solution of PZA was further diluted with glass distilled water, so as to obtain 50 ?l of diluted solution containing 200, 500, 1000, 2000, 4000 and 5000 ng respectively. The stock solution of IS was diluted with glass distilled water to obtain a concentration of 5000 ng per 50 ?l aliquot.
Calibration and sample preparation: For calibration curve, 50 ?l aliquots of working standard solutions of PZA corresponding to 200, 500, 1000, 2000, 4000 and 5000 ng concentration were pipetted into 15 ml glass tubes. To this 50 ?l of the internal standard solution (containing 5000 ng of NIA), 0.2 ml plasma were added, vortexed and left at room temperature for 5 min. An aliquot of 0.2 ml of 0.7 perchloric acid was added to each tube and vortexed thoroughly. After centrifugation at 4000 rpm for 10 min, 0.2 ml of the upper phase was removed and neutralized with 40 ?l of 1 M sodium hydroxide. Ten ?l of these neutralized samples were injected onto the liquid chromatograph.
Calibration curve specific to each volunteer was generated by spiking zero hour plasma with standard solutions and internal standard solution. The study samples were spiked with internal standard solution and analysed. As the absorption coefficient of internal standard (NIA) is 2/3 of PZA, it was necessary to use this (5000 ng) concentration to have adequate peak area ratio of PZANIA in the concentration of the PZA (range 200-500 ng).
Human studies: Human pilot studies were carried out in two overnight fasted healthy male volunteers. One received a PZA-Ciba 500 mg tablet orally with 150200 ml water. The other received 10 ml of PZA-Ciba syrup; residual syrup was rinsed with water and administered. Both volunteers received 500 mg of PZA. One volunteer was aged 20 years and weighed 62 kg; the other was 19 years old and weighed 59.5 kg. A standard breakfast was given after 2 hours and lunch after 4 hours of administration of drug. The blood samples were collected at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 34 and 48 hours post-administration in heparinized disposable syringes using sterile disposable needles. Plasma was separated by centrifugation, frozen and stored at -200 C until analysis.
Chromatography: As shown in [Figure:1], under the chromatographic conditions described, PZA and NIA- the internal standard were well resolved on the reversed-phase column, eluting at 5.2 and 6.0 minutes respectively and there was no interference from the endogenous components present in the plasma samples.
It was also observed that rifampicin was not eluted from the column: pyrazinoic acid, the major metabolite of PZA was not retained on the column; isoniazid and its major metabolite acetylisoniazicl were eluted at 4.0 min and 3.2 min respectively.
Linearity and reproducibility: The linearity and reproducibility of the method was established with human plasma spiked with PZA in the concentration range 2000 - 5000 ng/0.2 ml, and 5000 ng of IS (n= 6). The calibration curve generated in the above concentration range showed good linearity (intercept = - 0.01415; slope=0.00078; r=0.9997). [Table - 1] lists the reproducibility data obtained with six replicate analyses of the six concentrations.
Accuracy: The assay was applied to human plasma samples spiked with PZA in the range of 300 - 4500 ng/ 0.2 ml. Results presented in [Table - 2] indicate that the levels in above range can be estimated with good accuracy and precision with a coefficient & variation (CV) less than 5.0 %.
Precision: Both chromatographic and analytical precision with a day inter-assay (six replicate samples) and intra - assay (six injections of same sample) with same concentration assessed by CV is given in [Table - 3].
HPLC methods for the determination of PZA from biological fluids developed earlier involved an extraction of the drug,. HPLC method for determination of PZA from plasma eliminated the extraction of the drug but did not use an internal standard. The present method developed by us is quite simple and facile using NIA as an internal standard; the need for extraction of the drug is eliminated by using a simple precipitation method for sample preparation.
It is evident from [Figure:1] that PZA and NIA was well-resolved Supelco LC- 18 (1313) column without interference from the endogenous components. From the correlation coefficient (r = 0.9997), excellent linearity could be observed for assay of PZA in plasma. The good reproducibility/ accuracy of assay of PZA from is apparent from CV < 5.0 % [Table - 1] and [Table - 2]. The precision of within a day inter-assay and intra-assay as assessed by the CV [Table - 3], was also good.
This method has been tested to determine kinetics of PZA after a single oral dose of either PZA-Ciba 500 mg tablet or IPZA-Ciba syrup containing 500 mg active substance in two human volunteers is represented in [Figure:2].
Pyrazinamide was rapidly absorbed and attained C max of 8.98 Ug/ml and 12.81 ug/ml at 0.5 h and declined thereafter to a concentration of 0.66 ?g/ml at 34 h and 1.32 ?g/ml at 48 h with PZA-Ciba tablet and PZA-Ciba syrup respectively. The elimination half-lives calculated for PZA were 9.8 h and 10.9 h for PZA Ciba syrup respectively. The plasma AUC (0-34 h) values were 114.11 ?g/ml. h and 148.47 ?g/ml. h for tablet and syrup respectively.
The present HPLC method has advantages over the previously reported HPLC method. First, nicotinamide is used as internal standard, which corrects the sample preparation and injection errors. Second this method requires smaller plasma volume (0.2 ml) and third, the assay is facile, sensitive and selective such that pyrazinoic acid, the major metabolite and other anti -tuberculous drugs (Isoniazid. Rifampicin) do not interfere with the measurement of PZA. The major metabolite (pyrazinoic acid) did not retain on column and rifampicin did not elute under the chromatographic conditions. Isoniazicl and its major metabolite eluted at 4.0 min. and 3.2 min respectively.
[Table - 1], [Table - 2], [Table - 3]