Isoniazid (Figure 1
) is known for its antibacterial activity, which is caused by the inhibition of the biosynthesis of mycolic acid. It effectively kills bacteria that are actively growing in the body tissues with fluid content just within the first two weeks of treatment [1
]. Once administrated, isoniazid is well absorbed and distributed rapidly throughout the fluid part of the body. It is one of the drugs that is normally used in the treatment of tuberculosis and involves two phases. The initial stage lasts 2 months, and the main objective is to kill as many bacteria as possible. Due to the above-mentioned reason, in the first two-month period, rifampicin, pyrazinamide, isoniazid, and ethambutol are given to the patient. After the completion of the first stage, rifampicin and isoniazid are administered until the end of second stage of treatment which lasts another four months. Once ingested by the patient, isoniazid is primarily metabolized in the liver to acetyl isoniazid by acetyl transferase [2
]. The acetylation is controlled genetically, and it varies from patient to patient. Some of the patients are slow acetylators and some others are fast acetylators. Many researchers have reported that in fast acetylators, 90% of the drug is excreted as acetylisoniazid, resulting in short lasting effects of the drug which may lead to therapeutic failure [4
]. In slow acetylators, 67% of the drug is excreted in acetylated form resulting in a higher blood concentration of the parent drug, thus leaving patients prone to more severe side effects, such as peripheral neuropathy, which is the most common Central Nervous System (CNS)-related toxic effect [5
]. Due to its complex metabolic process, there is always a need for its monitorization in urine, especially to decide the proper dosage which normally varies from person to person depending on their metabolism, as it depends on highly-variable factors, like genetics, habits, health, physiological state, and the environment, among others.
Several methods for the detection and quantification of isoniazid from biological fluids and pharmaceuticals have been developed. These approaches use thin layer chromatography (TLC), high performance liquid chromatography) (HPLC), or liquid and gas chromatography coupled to mass spectrometry detectors (GC/MS, LC/MS) [6
]. However, these techniques require sophisticated and delicate instrumentation and trained laboratory staff and are relatively expensive in terms of maintenance and purchase. Apart from this, these methods are time-consuming, and require cumbersome pretreatment and a large amount of hazardous chemicals. Therefore, they are not suitable for routine clinical use [14
Micellar liquid chromatography (MLC), using a C18 column and the anionic surfactant sodium dodecyl sulfate, is a reverse-phase liquid chromatographic subtechnique that has great potential to overcome the above-mentioned problems [14
]. The major advantage of this technique is that it does not require any pretreatment for biological samples, as proteins and other endogenous compounds are denatured and solubilized by the micellar environment and can be harmlessly injected into the column. Besides, they are usually eluted at the front of the chromatogram, thus reducing the risk of overlapping with the analytes, even the barely retained ones [15
]. Many drugs, such as antitumorals, antiepileptics, antimalarials, antibiotics, and psychoactive compounds, in biological fluids, such as plasma, serum, and urine have already been successfully analyzed by MLC [16
]. The experimental protocol is expedited to a simple dilution, usually 1/5 v
, filtration, and direct injection in the column. The drugs are resolved from the matrix and endogenous compounds using a hybrid micellar mobile phase running under isocratic mode within a reasonable time period.
The aim of the work is to develop a simple, reliable, cost-effective, and rapid method for the determination of isoniazid in urine that can easily be used for the regular monitorization of isoniazid in tuberculosis patients in hospitals. It is validated by the Guidelines of the European Medicines Agency in order to verify its analytical performances, and applied to incurred samples [21
2.1. Chemical and Reagents
Isoniazid (purity >99.0%) was purchased from Merck (Darmstadt, Germany). Sodium dodecyl sulphate (SDS, 99% purity), sodium dihydrogen phosphate, and sodium acetate (analytical grade) were purchased from Himedia Laboratories Private Limited (Mumbai, India). Hydrochloric acid, sodium hydroxide, and HPLC grade 1-propanol were provided by Rankem, RFCL Limited (New Delhi, India). All the solutions were filtered through 0.45 µm nylon membrane filters from Micron Separation (Westboro, MA, USA). An ultrasonic bath (Model Ultrasons-H; Selecta, Barcelona, Spain) was used to achieve the solubilization of the solids. Ultrapure water was in-lab elaborated from deionized water using an Ultrapure water generator device, Simplicity UV (Millipore S.A.S., Molsheim, France). This water was used to prepare the aqueous solutions.
2.2. Apparatus and Instrumentation
The pH of the mobile phase was measured by using a digital pH meter pH-102/103 Contech, Instruments Limited, (Mumbai, India). The analytical weighing machine used was from Mettler Toledo India Private Limited (Mumbai, India).
Chromatographic analyses were performed on Shimadzu Prominence HPLC System, Shimadzu Corporation, (Kyoto, Japan) equipped with an isocratic pump LC-20 AT, an autosampler SIL-20AC, and a diode array detector SPD-M20 A (190–800 nm). The column used for the analysis was a SPHER-100 C18
100A (250 mm × 4.6 mm × 5 µm particle size) from Princeton Chromatography INC (Cranbury, NJ, USA). The mobile phase was an aqueous solution of 0.15 M SDS–12.5% 1-propanol–0.01 M Na2
0.01 M buffered at pH 7, running at 1 mL/min under isocratic mode at room temperature. The injection volume and the absorbance detection wavelength were 20 μL and 265 nm, respectively. All injected solutions, either standard of diluted samples, had been previously filtered through 0.45-μm nylon membrane filters (Micron Separations, Westboro, MA, USA) by pushing with a 3-mL syringe. The processed samples were thrown away after injection. The special care required for the HPLC system when working with micellar mobile phases is detailed in [23
A personal computer in which Shimadzu LC Solution software version 1.22 SP1 was installed was used to control the instrumentation, register the signal, and determine the main chromatographic parameters: retention time (tR
), retention factor (k
), efficiency (number of theoretical plates, N), and the asymmetry (B/A). These parameters were calculated as indicated in [24
2.3. Preparation of Solutions
Mobile phases were prepared by accurately weighing the proper amount of SDS and sodium dihydrogen phosphate salt, which were then dissolved in HPLC grade water with the aid of a magnetic stirrer. The pH was adjusted by adding drops of NaOH (0.1 M) or HCl (0.1 M). Thereafter, the organic solvent was introduced to reach the desired proportion, and then the volumetric flask was filled up with water, ultrasonicated for 5 min, and filtered through a through 0.45-μm nylon membrane filters (Micron Separations, Westboro, MA, USA) with the help of a vacuum pump.
Standard stock solution of isoniazid 100 µg/mL−1 was prepared by dissolving it in a small amount of water, following by ultrasonication (5 min). Working solutions were made by successive dilutions in the mobile phase.
2.4. Sample Collection and Processing
Urine samples from tuberculosis patients and healthy volunteers (taking no medications) were collected in glass tubes and stored at −20 °C, after consent regulated by the Ethical Committee of the Sagar’s Hospital (ethics approval code 2018-ICMR-001027, approved by the Indian Council for Medical Research). The investigations were carried out following the ethical rules of the Declaration of Helsinki 1975, revised in 2013. For confidentiality reasons, no personal or clinical information about the patients or the healthy volunteers (except that indicated below) was provided from the Hospital. The laboratory will not transmit any information to other institutions beyond that included in the publication and will destroy all of the urine samples and chromatogram files one year after publication of the paper.
Urine from healthy volunteers was drug-free. A matrix-matched blank sample was constructed by mixing equivalent volumes from six volunteers—three men and three women, including one individual in their twenties, another in their thirties and the last one in their forties for each gender. This sample was used as “blank sample” throughout the entire research.
Before being injected into the HPLC system, the preserved aliquots were brought to room temperature and 1/5 diluted with the optimized mobile phase without further pretreatment [14
]. For spiked samples, the appropriate volume of a working standard solution was added before dilution.