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Partial Validation of Ultra Performance Liquid Chromatography Method for Quantification of Isoniazid-Pyrazinamide
in Human Samples
Vycke Yunivita1,2*, Triana Nurul Meirina2, Atu Purnama Dewi2, Harold Eka Atmaja2, Rovina Ruslami1
1 Division of Pharmacology and Therapy, Department of Biomedical Sciences, Faculty of Medicine Universitas Padjadjaran, Jl. Prof. Eyckman No. 38, Sukajadi, Bandung, Indonesia
2 Pharmacokinetic Laboratory, Faculty of Medicine Universitas Padjadjaran, Bandung, Indonesia
*Corresponding email: v.yunivita@unpad.ac.id
Received 21 June 2021; Accepted 05 November 2021
ABSTRACT
Isoniazid (INH) and pyrazinamide (PZA) are first-line drugs on the treatment of meningitis of tuberculosis, in addition to rifampicin. The use of this drugs will determine the success of therapy to kill Mycobacterium tuberculosis in brain tissue that is difficult to penetrate by other drugs. Therefore, it is necessary to conduct research and monitor the use of this drug in plasma and cerebrospinal fluid (CSF) patients with TBM. This study aimed to determine the method of analysis of INH and PZA using Ultra Performance Liquid Chromatography Ultra Violet (UPLC-UV). The samples were taken from TBM patients who taken INH-PZA and others besides TBM patients who did not take INH-PZA. The analytic method carried out includes a comparison of the results of the analysis method for measuring levels of standard INH-PZA and INH-PZA in plasma and CSF samples. Analysis of INH-PZA in plasma and CSF can be performed using UPLC with UV detector, at least 100 uL plasma or CSF samples volume, with reproducible and accurate results.
Keywords: CSF, human plasma, isoniazid, pyrazinamide, UPLC
INTRODUCTION
Tuberculosis meningitis TB (TBM) is the most severe form of tuberculosis (TB) with a very high mortality and severe disability in more than half of living patients despite antituberculosis chemotherapy. With treatment using current therapeutic regimens, mortality is still high (50%), and occurs primarily in the first 2 weeks of treatment. Rifampicin (RIF), isoniazid (INH) and pyrazinamide (PZA) are antimycobacterial chemotherapeutic drugs known to inhibit tubercle bacilli both intracellularly and extracellularly [1-3]. These regimen are the backbone medications in the treatment of TB, include TBM [4]. These regimen be required to crossing the blood-brain barrier (BBB) or blood-cerebrospinal fluid-barrier (BCSFB) [1]. Due to varying TB dose penetrating across the BCSFB and BBB, monitoring drug levels in the cerebrospinal fluid and in the blood were one of the efforts to evaluate and optimize the use of existing anti-TB drugs for TBM patients.
Several methods for determination of one or more of the anti-TB drugs in plasma or serum are available [5-6]. Analysis of rifampicin in plasma and CSF has already been developed
[7]. However simultaneous determination of INH and PZA in plasma and CSF using ultra- performance liquid chromatography have not been reported.
INH is hydrazide isonicotinic acid which is a molecule with a mass of 137 and is soluble in water. In vitro, INH can inhibit the growth of mycobacteria at a concentration of 0.2 ug/ml obtained from oral use of 5 mg/kg/day. INH diffuses into all body fluids and tissues, concentrations in the cerebrospinal fluid (CSF) range from 20-100%. Pyrazinamide is stable, water-soluble nicotinamide. The use of a dose of PZA 25 mg/kg/day produces a concentration in the blood of 30-50 ug/ml and is distributed throughout the body, including the meninges [4].
In vitro studies established that both PZA and INH exhibit a concentration-dependent core.
Poor patient results were related with short levels of INH or PZA. So individuation of INH and PZA portions supported on their blood concentration and CSF could be helpful ini optimizing the drug remedy for some patients with poor response [5].
EXPERIMENT
Chemicals and instrumentation
INH and PZA standards used are USP products. Other reagents such as Methanol, Phosphoric Acid, Ethyl Acetate, 1-butanol, Potassium Dihydrogen Phosphate, Disodium Hydrogen Phosphate Dihydrate, and Ammonium Acetate were obtained from Merck Chemical, while Nicotinamide and sodium chloride were obtained from Sigma. The tool used for analysis in Ultra Performance Liquid Chromatography (UPLC) using UV detector. The separation was carried out with an HSS-T3 C18 (Waters) column using a gradient of acetate and methanol buffered mobile phases. Detection was carried out at a wavelength of 254 nm and a column temperature of 320C.
The plasma samples were taken from whole blood of subjects who taken and not taken INH-PZA on the therapy in heparin tube and must be centrifuged in 3000 rpm for 5 minutes.
The CSF samples were taken directly from subjects when lumbar puncture and collected in plastic tube. Samples were put in freezer in 800C to make it stable until the validation time.
Procedure reaction Analysis Method
A total of 100 uL of the samples was put into a conical glass tube that already contained 50 uL of nicotinamide standard internal solution and 1 ml of ethyl Acetate/butanol solution (70:30). Close the tube than vortex at 1800 rpm for 1 minute. Continue by centrifuging at 4300 g for 5 minutes. Pipette the top layer into the labelled micro-tube. Add 200 uL of 30 mM phosphoric acid. Vortex again at 2000 rpm for 30 seconds and then centrifuged at 4300 g for 5 minutes. Pipette 160 uL of the bottom layer into the autosampler vial which already contains 50 uL of 0.5 M Ammonium Acetate and then injects into the apparatus. The study was approved by the ethical review board of the Medical Faculty of Universitas Padjadjaran, Bandung, Indonesia (N0.41/UN6.C1.3.2/KEPK/PN/2015).
Validation Parameters
Several parameters tested in validating an analytical method are described and defined as
patients not on TB drug treatment were extracted and analyzed to see if any endogenous substances could interfere with the calculated response of INH and PZA. Observations were made by comparing them to the lower limit of the standard curve of the two analytes.
2. Accuracy and Precision
In order to validate an analytical method, intra-day and inter-day accuracy calculations were carried out at certain concentration variations of the analyte. The upper and lower limit standard solutions, as well as the internal control solutions (high, medium, low), were used as samples that represented the entire boundary of the standard curve. The analysis was carried out by repeating the sample 5 times from each concentration level which was calculated against the standard curve. To calculate inter-day precision and accuracy, intra- day testing was repeated on 3 different days.
3. Recovery (Recovery)
The recovery test was carried out by comparing the extraction results from standard solutions of INH and PZA spiked in plasma and cerebrospinal fluid with standard solutions made in solvents without extraction.
4. Stability
The stability test consisted of stability test in autosampler (storage after 25 and 48 hours), stability at room temperature (0, 2, 4, and 6 hours), and freeze-thaw stability. The sample used is an internal control solution consisting of 3 different concentrations (high, medium, low).
5. Detection Limit and Quantity Limit
The detection limit is the smallest amount of analyte in the sample that can be detected that still gives a significant response compared to the blank. The detection limit is a limit test parameter. The quantitation limit is a parameter in microscopic analysis and is defined as the smallest quantity of analyte in the sample that can still meet the criteria in terms of accuracy and precision.
RESULT AND DISCUSSION
Validation of an analytical method is an act of evaluating certain parameters based on laboratory experiments to prove that these parameters meet the requirements for their use.
Usually used as quality assurance when an analytical method is used to analyse a sample quantitatively.
1. Selectivity (Specificity)
INH, PZA, and standard internal Nicotinamide peaks appeared at retention times of 5.06, respectively; 6.89; and 7.76 minutes with a total injection time of 11 minutes. In this selectivity test, blank plasma is extracted and injected to see whether there is interference from the endogenous matrix in the plasma. The chromatogram results showed that no other peaks were found at the same retention time for the three analytes. This shows that the extraction method is selectively able to separate the three analytes to be analyzed well.
Comparison of the chromatograms of the three analytes can be seen in Figure 1.
Figure 1. Retention time for INH, PZA, and Nicotinamide (IS)
2. Precision and Accuracy
The precision value for isoniazid is in the range of 99.6% and 109.0% for plasma and 97.0%
and 102.9% for CSF dependent on standard concentration level, while pyrazinamide is in the range of 99.8% and 104.8% for plasma and 85.6% and 95.5% for CSF dependent on standard concentration level. To calculate the accuracy of inter-day and intra-day used coefficient of variation value of each analysis. The intra- and inter-day coefficient of variation for isoniazid were less than 7,94% over the 0.15 - 15 mg/L for plasma and less than 8.06% over the 0.15 - 15 mg/L for CSF concentration range. The intra- and inter-day coefficient of variation for pyrazinamide were less than 4,89% over the 0.2 - 60 mg/L for plasma and less than 6.6% over the 0.2 - 60 mg/L for CSF concentration range. The results obtained for both INH and PZA were within the acceptable limits specified by the Food Drug Administration (FDA) in the bioanalytic method validation guide.[8] The precision and accuracy data for the analysis of INH and PZA in cerebrospinal fluid can be seen in Table 1.
Table 1. Accuracy and Precision Intra and Inter-day
Drugs QC H QC M QC L
Accuracy (%)
Precision (RSD%)
Accuracy (%)
Precision (RSD%)
Accuracy (%)
Precision (RSD%) Intra-
day
Inter- day
Intra- day
Inter- day
Intra- day
Inter- day Plasma
INH 7.02 103.6 106.1 7.22 106.4 109.0 6.69 108.2 105.3
PZA 1.93 101.3 102.8 2.68 101.0 100.3 2.58 99.8 100.1
CSF
INH 2.86 100.1 99.4 3.45 97.5 97.1 8.06 97.1 99.1
3. Recovery
The percent recovery for INH and PZA was calculated by comparing the peak height of the extracted sample from each standard solution in plasma/cerebrospinal fluid and the peak in the solvent (acetonitrile 38%) without extraction. From the calculation results, the recovery value in plasma is 70.6% for INH and 17.2% for PZA, meanwhile the recovery value in CSF is 64.2% for INH and 18.3% for PZA. Although the PZA recovery value is relatively low, this result is still accepted, because both the precision and accuracy test results have values that are within the specified limits.
4. Stability
a) Autosampler Stability
The stability of the samples stored in the autosampler was tested after 25 and 48 hours of storage. The sample used for the autosampler stability test is the same sample as the injection sample for the calculation of analyte intra-day accuracy. The average value of the coefficient of variation obtained from the calculation results for this autosampler stability test is 5.73% for INH and 4.25% for PZA with an average precision ratio of the first day injection value of 96.5% for INH and 100.0% for PZA. Stability of the autosampler for PZA showed a relatively low number, however, the comparison of the recovery value with the initial value at 0 hour did not show a significant difference so that both were still said to be stable up to 48 hours of storage in the autosampler. INH and PZA autosampler stability data can be seen in Table 5.
b) Room Temperature Stability
The stability of the sample stored at room temperature was then extracted and injected into the device. Room temperature stability test was carried out at intervals of 2 hours for 6 hours. The samples used included 3 concentrations of high, medium, and low internal control in cerebrospinal fluid. The range of values obtained for accuracy is 5.2 to 7.1% and precision is 98.1 to 117.5% for INH while PZA has an accuracy value of 2.5 to 3.8% and a precision of 99.1 to 110 ,4. The results obtained showed that INH and PZA in cerebrospinal fluid were stable at room temperature for a storage time of 6 hours. The stability data of INH and PZA at room temperature can be seen in Table 3.
c) Freeze-thaw stability
Freeze-thaw stability test is intended to determine the stability of the sample when it is repeatedly thawed and then refrozen for analysis purposes. This test was carried out in 3 cycles, namely the first thawing on day 0, then refrozen for a minimum of 12 hours, after that it was thawed and refrozen. On the next day, the samples were thawed and tested. The results obtained show that both INH and PZA in
cerebrospinal fluid were stable after 3 thawing. The test results for freeze-thaw stability can be seen in Table 3.
Table 3. Summary of the validation results of various stability indicating parameters
Component Plasma CSF
Parameter Level INH PZA INH PZA
Freeze/thaw 3x QC H 104.2% 99.2% 98.2% 101.2%
QC M 98.2% 98.9% 90.8% 105.5%
QC L 90.2% 95.6% 93.5% 109.4%
Autosampler, 48h QC H 99.4% 114.2% 97.4% 91.4%
QC M 106.4% 102.1% 93.7% 84.8%
QC L 113.0% 101.7% 92.5% 84.5%
Freezer -80°C 6 months 12 months 6 months 6 months
Stock solution in water
(-80°C) 12 months 12 months
5. Detection Limit and Quantity Limit
The quantitation limit of this analytical method is 0.15 mg/L for INH and 0.20 mg/L for PZA, with the understanding that the concentration value below this limit cannot be accounted for in terms of accuracy or precision. While the detection limit value is calculated based on the ratio of the peak height of the lowest concentration of each analyte to the peak of the disturbance multiplied by the concentration of the quantitation limit. The calculation results show that the detection limit value for INH is 0.07 mg/L and 0.10 mg/L for PZA. In another report, the analytical method used to analyze INH in CSF also uses a chromatographic method with a UV detector, but when compared between the existing method and the method developed in the FKUP pharmacokinetic laboratory, this has a difference in the lower limit value which is smaller, namely 0.15 mg/L with a tool detection limit value of 0.067 mg/L. In addition, the use of Ultra Performance Liquid Chromatography (UPLC) compared to HPLC clearly has advantages in terms of the use of fewer reagents and faster elution time so that overall the use of these tools and methods is considered more profitable.[9]
CONCLUSION
Analysis of isoniazid and pyrazinamide in the plasma and in the cerebrospinal fluid can be performed using UPLC-UV. Liquid-liquid extraction to obtain both analytes using Nicotinamide as an internal standard is relatively easy and fast, besides that the sample volume used for analysis is only 100 uL, making it suitable for clinical pharmacokinetic studies. The validation results show that the analytical method for these two analytes in plasma and
All the author worked collectively to develop the methods described in this report. No conflict of interest associated with this work. VY received an internal Universitas Padjadjaran research funding with contract number 1427/UN6.3.1/LT/2020.
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