HPLC-based Method Research Articles

A Comparative Evaluation of Point-of-Care and Laboratory HbA1c Testing in Diabetes Care: An Indian Perspective.

This study aimed to assess the diagnostic performance of a point-of-care (POC) glycated hemoglobin (HbA1c) device in the Indian population against the standard laboratory (high-performance liquid chromatography {HPLC}) method for the effective management of diabetes in India. This study on the diagnostic accuracy of a POC HbA1c device involved 121 participants. These participants were categorized into two groups according to their HbA1c levels - one group with HbA1c values below 6.5% and another group with HbA1c values equal to or greater than 6.5%. The HbA1c levels in enrolled participants were estimated using both the POC device (HemoCueHbA1c 501; Ängelholm, Sweden: HemoCue AB) and the standard HPLC-based method. The level of agreement and concordance between the two test results were assessed by the Bland-Altman plot and Lin's concordance correlation coefficient. Sensitivity, specificity, diagnostic accuracy, positive likelihood, and negative likelihood ratios of POC-HbA1c device were assessed with a 6.5% HbA1c cut-off. The mean HbA1c values obtained by the two methods showed no statistically significant difference with a minimum effect size (Cohen's d = 0.035), indicating there was a negligible difference between these methods. The Bland-Altman plot revealed that most values were within acceptable limits (95% CI: -0.5 to 0.7) and Lin's concordance correlation coefficient showed strong agreement (p < 0.0001).The POC-HbA1c device demonstrated an area under the curve (AUC) of 0.991 (95% CI: 0.953-1.000) with sensitivity, specificity, diagnostic accuracy, positive likelihood, and negative likelihood ratios of 93.62%, 97.30%, 95.87%, 34.64 and 0.07, respectively, compared to the standard diagnostic assay. The diagnostic accuracy, sensitivity, and specificity demonstrated by the POC-HbA1c device to the standard HPLC method offers a viable and practical solution for diabetes management in India. Its ability to provide rapid and reliable results at the point of care can improve patient outcomes, reduce healthcare costs, and enhance access to diabetes care, especially in primary care, remote areas, and resource-limited settings of developing countries like India.

One-step N-Terminomics Based on Isolation of Protein N-Terminal Peptides From LysargiNase Digests by Tip-Based Strong Cation Exchange Chromatography

We have developed a one-step isolation method for protein N-terminal peptides from LysargiNase digests by pipette tip-based strong cation exchange (SCX) chromatography. This CHAMP-N (CHromatographic AMplification of Protein N-terminal peptides) method using disposable and parallel-processable SCX tips instead of conventional HPLC SCX columns facilitates simple, sensitive, reproducible, and high-throughput N-terminomic profiling without sacrificing the high identification numbers and selectivity achieved by the HPLC-based method. By applying the CHAMP-N method to HEK293T cells, we identified novel cleavage sites for signal and transit peptides and non-canonical translation initiation sites. Finally, for proteome-wide terminomics, we present a simple and comprehensive N- and C-terminomics platform employing three different tip-based approaches, including CHAMP-N, in which protease digestion and one-step isolation by tip LC are commonly used to achieve complementary terminome coverages.

Challenges in the Analytical Characterization of VLPs Through HPLC-Based Methods

Challenges in the Analytical Characterization of VLPs Through HPLC-Based Methods

A comprehensive measure of Golgi sphingolipid flux using NBD C6-ceramide: evaluation of sphingolipid inhibitors

Measurements of sphingolipid metabolism are most accurately performed by LC-MS. However, this technique is expensive, not widely accessible, and without the use of specific probes, it does not provide insight into metabolic flux through the pathway. Employing the fluorescent ceramide analogue NBD-C6-ceramide as a tracer in intact cells, we developed a comprehensive HPLC-based method that simultaneously measures the main nodes of ceramide metabolism in the Golgi. Hence, by quantifying the conversion of NBD-C6-ceramide to NBD-C6-sphingomyelin, NBD-C6-hexosylceramides, and NBD-C6-ceramide-1-phosphate (NBD-C1P), the activities of Golgi resident enzymes sphingomyelin synthase 1, glucosylceramide synthase, and ceramide kinase (CERK) could be measured simultaneously. Importantly, the detection of NBD-C1P allowed us to quantify CERK activity in cells, a usually difficult task. By applying this method, we evaluated the specificity of commonly used sphingolipid inhibitors and discovered that 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, which targets glucosylceramide synthase, and fenretinide (4HPR), an inhibitor for dihydroceramide desaturase, also suppress CERK activity. This study demonstrates the benefit of an expanded analysis of ceramide metabolism in the Golgi, and it provides a qualitative and easy-to-implement method.

Critical Secondary Metabolites Confer the Broad-Spectrum Pathogenic Fungi Resistance Property of a Marine-Originating Streptomyces sp. HNBCa1.

Obtaining a microorganism strain with a broad-spectrum resistance property and highly efficient antifungal activity is important to the biocontrol strategy. Herein, a marine Streptomyces sp. HNBCa1 demonstrated a broad-spectrum resistance to 17 tested crop pathogenic fungi and exhibited a high biocontrol efficiency against mango anthracnose and banana fusarium wilt. To uncover the critical bioactive secondary metabolites basis, genome assembly and annotation, metabolomic analysis, and a semipreparative HPLC-based activity-guide method were employed. Finally, geldanamycin and ectoine involved in codifferential secondary metabolites were also found to be related to biosynthetic gene clusters in the genome of HNBCa1. Reblastatin and geldanamycin were uncovered in response to broad-spectrum resistance to the 17 crop pathogenic fungi. Our results suggested that reblastatin and geldanamycin were critical to maintaining the broad-spectrum resistance property and highly efficient antifungal activity of HNBCa1, which could be further developed as a biological control agent to control crop fungal diseases.

Rapid HPLC method reveals dynamic shifts in coenzyme Q redox state

Ubiquinol or coenzyme Q (CoQ) is a lipid-soluble electron carrier in the respiratory chain and an electron acceptor for various enzymes in metabolic pathways that intersect at this cofactor hub in the mitochondrial inner membrane. The reduced form of CoQ is an antioxidant, which protects against lipid peroxidation. In this study, we have optimized a UV-detected HPLC method for CoQ analysis from biological materials, which involves a rapid single-step extraction into n-propanol followed by direct sample injection onto a column. Using this method, we have measured the oxidized, reduced and total CoQ pools, and monitored shifts in the CoQ redox status in response to cell culture conditions and bioenergetic perturbations. We find that hypoxia or sulfide exposure induces a reductive shift in the intracellular CoQ pool. The effect of hypoxia is however, rapidly reversed by exposure to ambient air. Interventions at different loci in the electron transport chain can induce sizeable redox shifts in the oxidative or reductive direction, depending on whether they are up- or downstream of complex III. We have also used this method to confirm that CoQ levels are higher and more reduced in murine heart versus brain. In summary, the availability of a convenient HPLC-based method described herein, will facilitate studies on CoQ redox dynamics in response to environmental, nutritional and endogenous alterations.

Development of ultra HPLC analytical method for pymetrozine residues in rice

Ultra HPLC-based analytical method analytical method was developed for the analysis of pymetrozine residues in different rice matrices. Chromatography separation was carried out in XR-ODS II: 150mm X 2.0mm, 5µm column in the mobile phase of water and methanol (60:40), and the resultant chromatograms were detected in a Photodiode Array (PDA). The retention time for pymetrozine neat standard and also matrix match standards was arrived at 4.00 min ± 0.5 min. The pymetrozine at the concentrations of 0.01, 0.05, 0.10, 0.25, 0.50, 0.75, and 1.00 µg/ml and the calibration curve of y = 2E+07x + 19188 obtained with a goodness of fit (R²) 0.995. The samples were extracted using the AOAC QuEChERS method with slight modifications. The harvest time residues for the different rice matrices were below the tolerance limit of pymetrozine. The residues of pymetrozine at 7, 15, and 30 days after the rice harvest in paddy seed, single-polished, double-polished rice, bran, and dehulled rice were not detected. From the developed analytical method, the residues of pymetrozine can be detected in different rice matrices.

4-Aldrithiol-based photometric assay for detection of methylthioalkylmalate synthase activity.

In Brassica plants, glucosinolates are a diverse class of natural products, of which aliphatic methionine-derived glucosinolates are the most abundant form. Their structural diversity comes from the elongation of some side-chains by up to 9 carbons, which, after the formation of the core glucosinolate structure, can undergo further chemical modifications. Methylthioalkylmalate synthase (MAMS) catalyzes the iterative elongation process for aliphatic methionine-derived glucosinolates. Most biochemical studies on MAMS have been performed using liquid chromatography/mass spectrometry (LC/MS)-based assays or high-performance liquid chromatography (HPLC)-based assays. The LC/MS- and HPLC-based methods are endpoint assays, which cannot be monitored in real time and require a laborious process for data collection. These analytical methods are inefficient for performing multiple enzymatic assays needed to determine steady-state kinetic parameters or for mechanistic evaluation of pH-dependence and kinetic isotope effect studies. Although the function of MAMS has long been defined, there is a gap in knowledge as it pertains to biochemical characterization of this plant enzyme. Part of this may be due to the lack of efficient methods that can be used for this type of research. This chapter describes a continuous photometric assay to track MAMS activity in real time using the 4-aldrithiol reagent for reaction detection.

RP HPLC-Based Method Development, Validation and Stability Indicating Assay of Levosulpiride in Bulk Drug Using Analytical Quality by Design Approach

RP HPLC-Based Method Development, Validation and Stability Indicating Assay of Levosulpiride in Bulk Drug Using Analytical Quality by Design Approach

Quantitative analytics for protein refolding states

Development and optimization of inclusion body (IB) refolding processes is often based on empirical strategies. Thus, generation of in-depth process understanding is crucial to shift towards more knowledge-driven approaches. Within this contribution the goal was to establish a systematic approach for the reliable quantification of process dynamics in protein refolding processes. Established offline- analytical tools like HPLC-based methods or photometric assays were analyzed regarding their specifications and consequent suitability to quantify protein states. An in-silico study was conducted to define requirements for state quantification by analyzing the influences of error, discrete sampling intervals and the pace of the reaction on signal quality. Refolding and aggregation reaction rates were calculated by finite difference approximation with errors propagated from the corresponding folding state. Application of the defined principles on two real Lactate dehydrogenase fed-batch refolding processes resulted in two individual sampling strategies. At reaction rates of up to 0.58 g h−1 description of the ongoing dynamics could be done accurately with high sampling intervals during the first few hours of processing as shown by comparison to a noise free simulation. The proposed method has the potential to be transferred to other proteins and to facilitate the development of model-based monitoring & control strategies.

Fast HPLC-based affinity method to determine capsid titer and full/empty ratio of adeno-associated viral vectors

Recombinant adeno-associated viruses (rAAVs) are promising gene delivery vectors in the emerging field of invivo gene therapies. To ensure their consistent quality during manufacturing and process development, multiple analytical techniques have been proposed for the characterization and quantification of rAAV capsids. Despite their indisputable capabilities for performing this task, current analytical methods are rather time-consuming, material intensive, complicated, and costly, restricting their suitability for process development in which time and sample throughput are severe constraints. To eliminate this bottleneck, we introduce here an affinity-based high-performance liquid chromatography method that allows the determination of the capsid titer and the full/empty ratio of rAAVs within less than 5min. By packing the commercially available AAVX affinity resin into small analytical columns, the rAAV fraction of diverse serotypes can be isolated from process-related impurities and analyzed by UV and fluorescence detection. As demonstrated by both method qualification data and side-by-side comparison with AAV enzyme-linked immunosorbent assay results for rAAV8 samples as well as by experiments using additional rAAV2, rAAV8, and rAAV9 constructs, our approach showed good performance, indicating its potential as a fast, simple and efficient tool for supporting the development of rAAV gene therapies.

B-034 Evaluation of the Analytical Performance of Sebia CAPI 3 HbA1c Assay

Abstract Background HbA1c has been the most important marker for glycemic control and for the diagnosis of diabetes mellitus. The NGSP has invested significant efforts in the standardization of the HbA1c assays, and several analytical methods have been developed. We evaluated the analytical performance of the Sebia CAPI 3 HbA1c assay, which is determined by capillary electrophoresis. Additionally, samples with and without hemoglobin variants are analyzed, and results are compared to two HPLC-based methods. Methods Control materials are used in the precision study, which are included in the Sebia CAPI 3 HbA1c kit (Lisses, France). Linearity study is assessed with the linearity materials from LGC Maine Standards (Cumberland Foreside, ME). Carryover is determined by the EP Evaluator (Colchester, VT) with total 21 aliquots of a high and a low HbA1c sample assayed in a specific order. Accuracy study is determined by whole blood patient samples without hemoglobin variants (n = 40) and with hemoglobin variants (n = 8, mostly with Hb S of 30–40%). Results from Sebia CAPI 3 are compared to the current laboratory method (BioRad Variant II Turbo) (Hercules, CA) and the TOSOH G8 (Tokyo, Japan). Both are HPLC-based methods. Results The within-run precision at a mean of 5.5% HbA1c has a CV at 1% (n = 24), and the between-run precision at the level of 5.5% and 8.7% HbA1c has a CV at 1% (n = 20) and 2% (n = 20), respectively. Linearity study demonstrates the analytical measuring range is between 3.1% and 20.8%, although each laboratory might establish its own clinical reportable range. Carryover is passed based on the automatically calculated error limit in EP Evaluator (3 times the SD of the Low-Low results). The accuracy study shows the Sebia results correlate well with percent bias at 2% for BioRad Variant II Turbo and 3% for TOSOH G8 in samples with (n = 8) and without (n = 40) hemoglobin variants. Conclusion The Sebia CAPI 3 HbA1c assay is based on capillary electrophoresis principle, which is a recognized method by NGSP. The assay has demonstrated good precision, a wide analytical measuring range and minimum carryover. The results are comparable to two HPLC-based methods and are well within the total allowable error of 6% by NGSP in samples with and without hemoglobin variant.

Phase-appropriate Application of Process Analytical Technology for Early Pharmaceutical Development of Oral Solid Dosage Forms-the Case Study of Uniformity Screening of Dosage Units and Blends.

Process analytical technology (PAT) in late-stage drug product development is typically used for real-time process monitoring, in-process control, and real-time release testing. In early research and development (R&D), PAT usage is limited as the manufacturing scale is relatively small with frequent changes and only a few batches are produced on an annual basis. However, process understanding is critical at early R&D in order to identify process and formulation boundaries, so PAT applications could be particularly useful in early-stage R&D. For oral solid dosage form, conventional HPLC-based content uniformity (CU) methods with sampling of 3 tablets per stratified sampling location in early R&D are typically not sufficient to identify these manufacturing process boundaries and temporal profile. Here, we report a screening CU method based on a multivariate model using transmission Raman spectroscopy (TRS) data on a phase-appropriate calibration set of only 16 tablets. This initial model was used for multiple pre-GMP development batches to provide critical information about blend uniformity and content uniformity (CU). In this work, the precision of the TRS method was evaluated; multiple spectral preprocessing approaches were compared regarding their effects on measurement precision as well as their ability to mitigate the photo bleaching effects during precision experiments. Overall, the TRS-based CU method was much faster than a traditional HPLC-based method allowing a much larger number of tablets to be screened. This larger number of analyzed tablets enabled the processes boundaries and temporal changes in CU to be identified while providing proper statistical assurance on product quality.

Application of a Quantitative Proton Nuclear Magnetic Resonance Method for the Determination of Protopine in Radix Dactylicapnotis

Background: The Bai ethnologic herb Radix Dactylicapnotis, the root and tuber of Dactylicapnos scandens (Papaveraceae), is used for clearing heat, relieving pain, and achieving hemostasis and antihypertensive effects. Objective: The study aimed to develop a quantitative method for determining the protopine content in Radix Dactylicapnotis by using proton nuclear magnetic resonance (1H NMR) spectroscopy. Methods: The deuterium solvent, internal standard, and NMR parameters were optimized. The quantitative method was validated by linearity, precision, accuracy, repeatability, and stability, as well as limit-of-detection (LOD) and limit-of-quantitation (LOQ) assays. Results: A mixture solution consisting of 500 μL of DMSO-d6 and 20 μL of D2O enabled satisfactory separation of the signals to be integrated into the 1H NMR spectrum. Trimethyl benzene-1,3,5- tricarboxylate (TMBT) was selected as an internal standard. The integration of δ 6.05-6.08 corresponding to OCH2O was selected to quantify protopine. The developed quantitative method was found to be precise and accurate and to exhibit excellent linearity and range. The protopine content in Radix Dactylicapnotis could be quantified accurately using the featured signal. Conclusion: This is the first study to report quantitative 1H NMR determination of protopine in Radix Dactylicapnotis. The study results indicate that quantitative 1H NMR represents a feasible alternative to HPLC-based methods for the quantitation of protopine in Radix Dactylicapnotis, and is suitable for the quality control of Radix Dactylicapnotis.

A robust, viable, and resource sparing HPLC-based logP method applied to common drugs

Reliable, experimentally determined partition coefficient P (logP) for most drugs are often unavailable in the literature. Many values are from in silico predictions and may not accurately reflect drug lipophilicity.In this study, a robust, viable, and resource sparing method to measure logP was developed using reverse phase high performance liquid chromatography (RP-HPLC). The logP of twelve common drugs was measured using calibration curves at pH 6 and 9 that were created using reference standards with well-established logP.The HPLC method reported here can be used for high throughput estimation of logP of commonly used drugs. LogP values here showed general agreement with the other few HPLC-based literature logP values available. Additionally, the HPLC-based logP values found here agreed partially with literature logP values found using other methodologies (±10%). However, there was no strong agreement since there are few experimentally determined literature logP values. This paper shows a facile method to estimate logP without using octanol or computational approaches.This method has excellent promise to provide reliable logP values of commonly used drugs available in literature. A larger pool of reliable logP values of commonly drugs has promise to improve quality of medicinal chemistry and pharmacokinetic (PK) models.

Optimization of aqueous extraction of henna leaves (Lawsonia inermis L.) and evaluation of biological activity by HPLC-based profiling and molecular docking techniques

Due to the antimicrobial capabilities of henna leaf extract (Lawsonia inermis L.), this study intends to identify environmentally friendly ideal conditions for extracting bioactive compounds from henna leaf powder. For this purpose, the maceration method with a 100% aqueous solvent was selected for further analysis. Optimization of the extraction method was carried out by central composite design (CCD) protocol as a subset of Response Surface Methodology (RSM). The temperature of 40–90 °C, extraction time of 2–24 h, and the solvent-plant weight ratio ranging from 10 to 100 mL/mg were all considered independent variables. Also, the amount of lawsone, as an active component, and the total phenolic content were tracked as responses. Finally, a yield of 30.59% was achieved under the best extraction condition at 40 °C, 2 h, and a solvent-plant weight ratio of 1:100. The biological activity of an aqueous extract was evaluated using an HPLC-based activity profiling and TLC bioautography method. Peaks collected as fractions between 3 and 30 min were tested for antibacterial activity against Staphylococcus aureus ATCC 25923, and micro fractions from 5, 6, 7.5, 9, 10.5, 12, 13.5, 15, 19, and 30 min exhibited significant inhibitory effects. All spotted fractions on the TLC plate inhibited the growth of Escherichia coli ATCC 25922, except for micro fractions from 3, 18, and 21 min. Moreover, molecular docking analysis of quinones in the extract identified 3a, 4a-Dihydroxy-a-tetralone (C-2) as the most potent compound in interacting with bacteria sterol 14-demethylase protein, with docking scores of − 6.715 and − 7.196 kcal/mol, respectively.

Development of C1q Affinity Chromatography for the Study of C1q-IgG Interactions.

The classical complement system represents a central effector mechanism of Abs initiated by the binding of C1q to target bound IgG. Human C1q contains six heterotrimeric globular head groups that mediate IgG interaction, resulting in an avidity-driven binding event involving multiple IgG molecules binding a single C1q. Accordingly, surface bound IgG molecules are thought to assemble into noncovalent hexameric rings for optimal binding to the six-headed C1q. To study the C1q-Fc interaction of various Abs and screen for altered C1q binding mutants, we developed, to our knowledge, a novel HPLC-based method. Employing a single-chain form of C1q representing one C1q head group, our HPLC methodology was able to detect the interaction between the single-chain monomeric form of C1q and various ligands. We show that, despite a narrow window of specific binding owing to the low affinity of the monomeric C1q-IgG interaction, this approach clearly distinguished between IgG subclasses with established C1q binding properties. IgG3 displayed the strongest binding, followed by IgG1, with IgG2 and IgG4 showing the weakest binding. Fc mutants known to have increased C1q binding through oligomerization or enhanced C1q interaction showed greatly increased column retention, and IgG glycovariants displayed a consistent trend of increasing retention upon increasing galactosylation and sialylation. Furthermore, the column retention of IgG isotypes and glycovariants matches both the cell surface recruitment of C1q and complement-mediated cytotoxicity induced by each variant on an anti-CD20 Ab backbone. This methodology therefore provides a valuable tool for testing IgG Ab (glyco)variants for C1q binding, with clear relevance for therapeutic Ab development.

Development and Validation of a High-Performance Liquid Chromatography-Based Method for Catechin Isolated from the Leaves of Gambir (Uncaria gambir Roxb)

Gambir (Uncaria gambir Roxb) has long been thought to provide various health benefits due to the main secondary metabolite, catechin. The high catechin content makes gambir a strong antioxidant. The present study was conducted to develop and validate the quantification of catechin in gambir leaves by high-performance liquid chromatography (HPLC). The method was performed using a 100 mL sample with dilution. The separation process employed a Sunfire C18 (150 mm × 4.6 mm) column, the mobile phase was composed of solvent A: 0.03% trifluoroacetic acid in acetonitrile-water (5:95) and solvent B: 0.1% trifluoroacetic acid in acetonitrile at a 0.45 mL/min flow rate. The validation was conducted by measuring the accuracy, precision, linearity, limit of detection (LOD), limit of quantification (LOQ), and robustness. The results showed that the percent recovery was 98.30-99.82% with a precision value of 0.28% RSD, good linearity with a 0.9996 R-value, 2.7 µg/mL of LOD, and 8.3 µg/mL of LOQ. There was no significant difference between changes in the mobile phase of 4% (comparison of area and retention time), and 6% (comparison of area). However, the results also demonstrated that the mobile phase was not resistant to changes in the mobile phase of 6% in the ratio of retention time because of the shift in retention time that was faster than the catechin standard. The findings of this study reveal that this method satisfies the validation requirements for accuracy, precision, linearity, and robustness tests, therefore, the HPLC-based method can be applied to analyze catechin in gambir leaves.

A sensitive and economical method for simultaneous determination of D/L- amino acids profile in foods by HPLC-UV: Application in fermented and unfermented foods discrimination

This work described a sensitive and economical HPLC-UV method with FDAA derivatization to simultaneously detect 36 D/l-amino acids, which provides higher sensitivity and lower cost than other HPLC-based methods. It was validated for linearity range (8–1000 µmol/L), limits of detection (2.68–62.44 pmol/L), limits of quantification (2.93 to 208.13 pmol/L), intraday precision (0.30 % − 5.31 %), interday precision (1.96 % − 8.04 %) and accuracy (86.53 % − 121.46 %). This method was then applied in the determination of D/L- amino acids abundance in fermented and unfermented food materials and showed the characteristics of each type of foods. The method also demonstrated good performance in another application case for the discrimination of different types of food materials based on D/L- amino acids profile. It emphasizes the ability of the method to study the characteristics, distribution and abundance of d-amino acids in foods and their potential application in food quality control.

High-performance Liquid Chromatography based Methods for Determination of Antitubercular Drugs in Urine Samples: A Scoping Review

Introduction: Tuberculosis (TB) is a contagious disease and is one of the leading causes of death worldwide. TB is primarily caused by Mycobacterium Tuberculosis (MTB) and the percentage of Multi-Drug Resistance TB (MDR-TB) and Extensively Drug Resistance TB (XDR-TB) is increasing daily in many developing countries. Aim: To summarise different High-performance Liquid Chromatography (HPLC)-based methods for checking treatment adherence and drug monitoring of patients by estimating the amount of Anti-tubercular (Anti-TB) drugs in urine samples. Materials and Methods: The authors conducted a search and screened various databases (PubMed, Scopus, Web of Science, Google Scholar) using different keywords between April 2023 and June 2023. The authors included original research articles, clinical trials, and observational studies that focused on estimating anti-TB drugs in urine samples using HPLC. The authors excluded articles that employed methods other than HPLC for drug estimation in urine samples. A total of 13 articles were included in this review. Results: The authors identified 296 articles from different electronic databases and four articles from other sources (Google Scholar, ResearchGate, etc.). Ultimately, 13 articles were included describing HPLC-based methods for determining anti-TB drugs in urine samples. Data was extracted focusing on mobile phases and sample preparation/extraction procedures. Two articles of 2004 and 2014 reported simple mobile phases and sample preparation methods for estimating rifampicin and isoniazid. Additionally, articles published in the last five years have employed simple mobile phases with minimal or no extraction procedures. Conclusion: The present review summarises various HPLCbased methods reported in the literature, as it is considered the gold standard method for checking treatment adherence in TB patients. Urine samples were chosen for ease of collection, particularly from patients of different age groups, including the paediatric population. This review highlights the need for more HPLC-based methods with simple mobile phases and extraction procedures for early detection of anti-TB drugs in resource-poor settings.