Baclofen impurities: Facile synthesis and novel environmentally benign chromatographic method for their simultaneous determination in baclofen

An efficient, economic and high yielding method was described for the synthesis of baclofen (BAC) pharmacopoeial impurities (impurity A and impurity B) which can be used for gram‐scale synthesis. Furthermore, a novel ecofriendly thin‐layer chromatographic TLC–densitometric method was established and validated for the determination of BAC and its synthesized impurities. The developed TLC–densitometric method is based on the chromatographic separation using TLC plates (60 F₂₅₄) using a green mobile phase of ethyl acetate–methanol–ammonia solution, 33% (8:2:0.1, by volume) with UV scanning at 220 nm. The proposed method was validated with respect to International Conference on Harmonization guidelines. The validated method was successfully applied for determination of BAC in pure form and in its commercial dosage form. Additionally, the greenness profile of the developed method was evaluated and compared with those of the reported chromatographic methods. The developed method was found to be superior to the published methods, being environmentally benign.     

Different stability‐indicating chromatographic methods for specific determination of paracetamol,dantrolene sodium,their toxic impurities and degradation products

A well‐known analgesic (paracetamol, PAR) and skeletal muscle relaxant [dantrolene sodium (DNS)] have been analyzed without interference from their toxic impurities and degradation products. The studied PAR impurities are the genotoxic and nephrotoxic p‐amino phenol (PAP) and the hepatotoxic and nephrotoxic chloroacetanilide, while 5‐(4‐nitrophenyl)‐2‐furaldehyde is reported to be a mutagenic and carcinogenic degradation product of DNS. The five studied components were determined and quantified by TLC–densitometric and RP‐HPLC methods. TLC–densitometry (method 1) used TLC silica gel and chloroform–ethyl acetate–acetic acid–triethylamine (7:3:0.5:0.05, by volume) as the mobile phase with UV scanning at 230 nm, while RP‐HPLC (method 2) was based on separation on a C₁₈ column using methanol–water (55:45, v/v pH 3 with aqueous formic acid) as mobile phase at 1 mL/min and detection at 230 nm. The developed methods were used for determination and quantification of the five studied components in different laboratory‐prepared mixtures. The were also applied for analysis of Dantrelax^® compound capsules where no interference among the studied components with each other or from excipients was observed. The methods were validated as per International Conference on Harmonization guidelines, and they compared favorably with the reported ones.     

Simultaneous HPTLC and RP-HPLC methods for determination of bumadizone in the presence of its alkaline-induced degradation product

Accurate, selective, sensitive and precise HPTLC‐densitometric and RP‐HPLC methods were developed and validated for determination of bumadizone calcium semi‐hydrate in the presence of its alkaline‐induced degradation product and in pharmaceutical formulation. Method A uses HPTLC‐densitometry, depending on separation and quantitation of bumadizone and its alkaline‐induced degradation product on TLC silica gel 60 F₂₅₄ plates, using hexane–ethyl acetate–glacial acetic acid (8:2:0.2, v/v/v) as a mobile phase followed by densitometric measurement of the bands at 240 nm. Method B comprises RP‐HPLC separation of bumadizone and its alkaline‐induced degradation product using a mobile phase consisting of methanol–water–acetonitrile (20:30:50, v/v/v) on a Phenomenex C₁₈ column at a flow‐rate of 2 mL/min and UV detection at 235 nm. The proposed methods were successfully applied to the analysis of bumadizone either in bulk powder or in pharmaceutical formulation without interference from other dosage form additives, and the results were statistically compared with the established method. Copyright © 2011 John Wiley & Sons, Ltd.     

Development and Validation of HPLC,TLC and Derivative Spectrophotometric Methods for the Analysis of Ezetimibe in the Presence of Alkaline Induced Degradation Products

Reversed phase‐high performance liquid chromatography (RP‐HPLC), thin layer chromatography (TLC) densitometry and first derivative spectrophotometry (1D) techniques are developed and validated as a stability‐indicating assay of ezetimibe in the presence of alkaline induced degradation products. RP‐HPLC method involves an isocratic elution on a Phenomenex Luna 5μ C₁₈ column using acetonitrile: water: glacial acetic acid (50:50:0.1 v/v/v) as a mobile phase at a flow rate of 1.5 mL/min. and a UV detector at 235 nm. TLC densitometric method is based on the difference in Rf‐values between the intact drug and its degradation products on aluminum‐packed silica gel 60 F₂₅₄ TLC plates as stationary phase with isopropanol: ammonia 33% (9:1 v/v) as a developing mobile phase. On the fluorescent plates, the spots were located by fluorescence quenching and the densitometric analysis was carried out at 250 nm. Derivative spectrophotometry, the zero‐crossing method, ezetimibe was determined using first derivative at 261 nm in the presence of its degradation products. Calibration graphs of the three suggested methods are linear in the concentration ranges 1–10 mcg/mL, 0.1–1 mg/mL and 1–16 mcg/mL with a mean percentage accuracy of 99.05 ± 0.54%, 99.46 ± 0.63% and 99.24 ± 0.82% of bulk powder, respectively. The three proposed methods were successfully applied for the determination of ezetimibe in raw material and pharmaceutical dosage form; the results were statistically analyzed and compared with those obtained by the reported method. Validation parameters were determined for linearity, accuracy and precision; selectivity and robustness and were assessed by applying the standard addition technique.     

Stability-Indicating Chromatographic Methods for Simultaneous Determination of Mosapride and Pantoprazole in Pharmaceutical Dosage Form and Plasma Samples

Simple, sensitive, selective, precise, and stability-indicating thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) methods for the determination of mosapride and pantoprazole in pharmaceutical tablets were developed and validated as per the International Conference on Harmonization guidelines. The TLC method employs aluminum TLC plates precoated with silica gel 60F₂₅₄ as the stationary phase and ethyl acetate/methanol/toluene (4:1:2, v/v/v) as the mobile phase to give compact spots for mosapride (R _f 0.73) and pantoprazole (R _f 0.45) separated from their degradation products; the chromatogram was scanned at 276 nm. The HPLC method utilizes a C18 column and a mobile phase consisting of acetonitrile/methanol/20 mM ammonium acetate (4:2:4, v/v/v) at a flow rate of 1.0 mL min^?1 for the separation of mosapride (t _R 11.4) and pantoprazole (t _R 4.4) from their degradation products. Quantitation was achieved with UV detection at 280 nm. The same HPLC method was successfully used in performing calibrations in lower concentration ranges for both drugs in human plasma using ezetimibe as internal standard. The methods were validated in terms of accuracy, precision, linearity, limits of detection, and limits of quantification. Mosapride and pantoprazole were exposed to acid hydrolysis and then analyzed by the proposed methods. As the methods could effectively separate the drugs from their degradation products, these techniques can be employed as stability-indicating methods that have been successively applied to pharmaceutical formulations without interference from the excipients. Moreover the HPLC method was successfully used in the determination of both drugs in spiked human plasma.     

Development and validation of a stability‐indicating assay including the isolation and characterization of degradation products of metaxalone by LC‐MS

A stability‐indicating reverse‐phase high‐performance liquid chromatography–mass spectrometric method was developed and validated for the assay of metaxalone through forced degradation under acidic, alkaline, photo, oxidative and peroxide stress conditions. Separation of degradation products was accomplished on a reverse‐phase Phenomenex C₁₈ (250 × 4.6 mm, 5 µm) column thermostated at 25°C using 10 mM aqueous ammonium acetate: methanol (35:65 v/v) as mobile phase in an isocratic mode of elution. The eluents were detected at 275 nm by photo diode array detector and mass detectors connected in series. Two unknown base hydrolysis products of metaxalone were identified and characterized as (a) methyl 3‐(3,5‐dimethylphenoxy)‐2‐hydroxypropylcarbamate and (b) 1‐(3,5‐dimethylphenoxy)‐3‐aminopropan‐2‐ol by MS, ¹H NMR and FTIR spectroscopy. The method was validated as per International Conference on Harmonization guidelines and metaxalone was selectively determined in presence of its degradation impurities, demonstrating its stability‐indicating nature. Copyright © 2013 John Wiley & Sons, Ltd.     

Validation of an ultra‐performance liquid chromatography–tandem mass spectrometry method for the determination of flecainide in human plasma and its clinical application

A simple and reproducible bioanalytical method for the determination of flecainide in human plasma was developed and validated using an ultra‐performance liquid chromatography with tandem mass spectrometry (UPLC‐MS/MS) to obtain higher sensitivity than the current available methods. After simple protein precipitation, flecainide and a stable isotope‐labeled internal standard (IS) were chromatographed on an Acquity UPLC BEH C₁₈ column (2.1 × 100 mm, 1.7 µm) with isocratic elution of mobile phase consisting of 45% methanol containing 0.1% formic acid at a flow rate 0.25 mL/min. Detection was performed in positive electrospray ionization by monitoring the selected ion transitions at m/z 415.4/301.1 for flecainide and m/z 419.4/305.1 for the IS. The method was validated according to current bioanalytical method validation guidelines. The calibration standard curve was linear from 2.5 to 1000 ng/mL using 0.1 mL of plasma. No significant interferences were detected in blank human plasma. Accuracy and precision in the intra‐ and inter‐batch reproducibility study were within acceptance criteria. Neither hemolysis effects nor matrix effects were observed. The UPLC‐MS/MS method developed was successfully applied to determine plasma flecainide concentrations to support clinical studies and incurred sample reanalysis also ensured the reproducibility of the method. Copyright © 2015 John Wiley & Sons, Ltd.     

Stability-Indicating Chromatographic Methods for Simultaneous Determination of Mosapride and Pantoprazole in Pharmaceutical Dosage Form and Plasma Samples

Simple, sensitive, selective, precise, and stability-indicating thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) methods for the determination of mosapride and pantoprazole in pharmaceutical tablets were developed and validated as per the International Conference on Harmonization guidelines. The TLC method employs aluminum TLC plates precoated with silica gel 60F₂₅₄ as the stationary phase and ethyl acetate/methanol/toluene (4:1:2, v/v/v) as the mobile phase to give compact spots for mosapride (R _f 0.73) and pantoprazole (R _f 0.45) separated from their degradation products; the chromatogram was scanned at 276 nm. The HPLC method utilizes a C18 column and a mobile phase consisting of acetonitrile/methanol/20 mM ammonium acetate (4:2:4, v/v/v) at a flow rate of 1.0 mL min⁻¹ for the separation of mosapride (t _R 11.4) and pantoprazole (t _R 4.4) from their degradation products. Quantitation was achieved with UV detection at 280 nm. The same HPLC method was successfully used in performing calibrations in lower concentration ranges for both drugs in human plasma using ezetimibe as internal standard. The methods were validated in terms of accuracy, precision, linearity, limits of detection, and limits of quantification. Mosapride and pantoprazole were exposed to acid hydrolysis and then analyzed by the proposed methods. As the methods could effectively separate the drugs from their degradation products, these techniques can be employed as stability-indicating methods that have been successively applied to pharmaceutical formulations without interference from the excipients. Moreover the HPLC method was successfully used in the determination of both drugs in spiked human plasma.

     

Resolution and isolation of enantiomers of (±)‐isoxsuprine using thin silica gel layers impregnated with l‐glutamic acid,comparison of separation of its diastereomers prepared with chiral derivatizing reagents having l‐amino acids as chiral auxiliaries

Thin silica gel layers impregnated with optically pure l ‐glutamic acid were used for direct resolution of enantiomers of (±)‐isoxsuprine in their native form. Three chiral derivatizing reagents, based on DFDNB moiety, were synthesized having l ‐alanine, l ‐valine and S‐benzyl‐l ‐cysteine as chiral auxiliaries. These were used to prepare diastereomers under microwave irradiation and conventional heating. The diastereomers were separated by reversed‐phase high‐performance liquid chromatography on a C₁₈ column with detection at 340 nm using gradient elution with mobile phase containing aqueous trifluoroacetic acid and acetonitrile in different compositions and by thin‐layer chromatography (TLC) on reversed phase (RP) C₁₈ plates. Diastereomers prepared with enantiomerically pure (+)‐isoxsuprine were used as standards for the determination of the elution order of diastereomers of (±)‐isoxsuprine. The elution order in the experimental study of RP‐TLC and RP‐HPLC supported the developed optimized structures of diastereomers based on density functional theory. The limit of detection was 0.1–0.09 µg/mL in TLC while it was in the range of 22–23 pg/mL in HPLC and 11–13 ng/mL in RP‐TLC for each enantiomer. The conditions of derivatization and chromatographic separation were optimized. The method was validated for accuracy, precision, limit of detection and limit of quantification. Copyright © 2014 John Wiley & Sons, Ltd.     

Comparison of Two Stability-Indicating Chromatographic Methods for the Determination of Mirabegron in Presence of Its Degradation Product

Mirabegron is a novel β₃-adrenoceptor agonist containing an amide group. It was subjected to stress conditions of acidic and alkaline hydrolyses. The hydrolytic degradation product was isolated and its structure was confirmed using mass and IR spectrometry. Two stability-indicating chromatographic methods have been proposed for the determination of mirabegron. TLC method was applied using silica gel as stationary phase and chloroform–methanol–ammonia (9.0:1.0:0.1 by volume) as the mobile phase, and chromatograms were scanned at 250 nm. Accurate determination of the drug was achieved over the concentration range of 2–12 μg per band. In addition, an isocratic HPLC method was developed on Agilent C18 column (150 mm × 4.5 mm I.D., particle size 5 µm) using ethanol-phosphate buffer pH 2.5 (30:70, by volume) as a mobile phase with flow rate of 1 mL min^?1.The intact drug was detected at 250 nm with running time less than 5 min. Mirabegron was determined accurately in a concentration range of 1–25 µg mL^?1. The proposed chromatographic methods were applied successfully for the assay of mirabegron in pharmaceutical dosage form and both methods were validated as per the International Conference on Harmonization guidelines and statistically compared with a reported gradient HPLC method.     

Stereoselective analysis of flecainide enantiomers using reversed‐phase liquid chromatography for assessing CYP2D6 activity

Stereoselective analyses of flecainide enantiomers were performed using reversed‐phase high‐performance liquid chromatography (HPLC) equipped with a polysaccharide‐based chiral column (Chiralpak AS‐RH) and fluorescence detector. Excitation and emission wavelengths were set at 300 and 370 nm, respectively. Flecainide enantiomers in serum and urine were extracted using diethyl ether. The mobile phase solution, comprising 0.1 m potassium hexafluorophosphate and acetonitrile (65:35, v/v), was pumped at a flow rate of 0.5 mL/min. The recoveries of flecainide enantiomers were greater than 94%, with the coefficients of variation (CVs) <6%. The calibration curves of flecainide enantiomers in serum and urine were linear in the concentration range 5–500 ng/mL and 0.75–15 µg/mL (r > 0.999), respectively. CVs in intra‐day and inter‐day assays were 1.8–5.8 and 3.4–7.5%, respectively. In a pharmacokinetic study, the ratios of (S)‐ to (R)‐flecainide (S/R ratio) in the area under the curve and the amount of flecainide enantiomers excreted in urine were lower in a subject carrying CYP2D6*10/*10 than in subjects carrying CYP2D6*1/*2. The S/R ratio of trough serum flecainide concentration ranged from 0.79 to 1.16 in patients receiving oral flecainide. The present HPLC method can be used to assess hepatic flecainide metabolism in a pharmacokinetic study and therapeutic drug monitoring. Copyright © 2014 John Wiley & Sons, Ltd.     

Chromatographic determination of sulfasalazine and its active metabolites: greenness assessment and application to spiked human plasma

Green TLC-densitometric and RP-HPLC methods were developed and validated for the determination of the active prodrug sulfasalazine (SZ), its active metabolite mesalazine (MZ) and the major active metabolite of mesalazine, N-acetyl-5-aminosalicylic acid (AS). In the developed TLC-densitometric method, chromatographic separation was carried out on TLC silica gel plates 60 F₂₅₄ using a developing system consisting of ethyl acetate–methanol–ammonia solution 33% (8:2.5:0.3, by volume) and then scanning the separated bands at 215 nm using hydrochlorothiazide as an internal standard with linearity ranges of 0.4–3, 0.4–2.4 and 0.3–2 for SZ, MZ and AS, respectively. The developed RP-HPLC method depended on chromatographic separation using a C₁₈ column with a solvent mixture of methanol–aqueous acetic acid solution (pH 5) as a mobile phase with gradient elution mode and UV scanning at 243 nm using pyrazinamide as internal standard with linearity ranges of 5–50, 5–40, and 3–20 for SZ, MZ and AS, respectively. US Food and Drug Administration guidelines were followed during validation of the methods. The greenness of the developed methods was estimated using the greenness profile and the Eco-Scale approach. Both methods passed the four quadrants of the greenness profile and had Eco-Scale score ˃75, thus they were considered to be green according to these approaches.     

A degradation study of cefepime hydrochloride in solutions under various stress conditions by TLC–densitometry

A rapid, accurate and sensitive thin‐layer chromatography (TLC) method with densitometric detection has been developed and validated for the determination of cefepime in pharmaceuticals. Chromatographic separation was achieved on a silica gel TLC F₂₅₄ plates with a mobile phase consisting of ethanol–2‐propanol–glacial acetic acid 99.5%–water (4:4:1:3, v/v). Densitometric detection was carried out at wavelength of 266 nm in reflectance/absorbance mode. The validation of the method was found to be satisfactory with high accuracy (from 99.24 to 101.37%) and precision (RSD from 0.06 to 0.36%). Additionally, the stability of cefepime in solution was investigated, including the effect of pH, temperature and incubation time. Favorable retention parameters (R_f, R_s, α) were obtained under the developed conditions, which guaranteed good separation of the studied components. The degradation process of cefepime hydrochloride was described by kinetic and thermodynamic parameters (k, t_0.1, t_0.5 and E_a). Moreover, the chemical properties of degradation products were characterized by the R_f values, absorption spectra, HPLC‐MS/MS and TLC‐densitometry analysis. As the method could effectively separate the active substance from its main degradation product (1‐methylpyrrolidine), it can be employed as a method to indicate the stability of this drug. Copyright © 2014 John Wiley & Sons, Ltd.     

A validated stability‐indicating ultra performance liquid chromatography method for the determination of potential process‐related impurities in eplerenone

A simple, sensitive, and accurate stability‐indicating analytical method has been developed and validated using ultra high performance liquid chromatography. The developed method is used to evaluate the related substances of eplerenone (EP). The degradation behavior of EP under stress conditions was determined, and the major degradants were identified by ultra high performance liquid chromatography with tandem mass spectrometry. The chromatographic conditions were optimized using an impurity‐spiked solution, and the samples, generated from forced degradation studies. The resolution of EP, its potential impurities, and its degradation products was performed on a Waters UPLC BEH C₁₈ column (50 × 2.1 mm, 1.7 μm) by linear gradient elution using a mobile phase consisting of 10 mmol/L ammonium acetate adjusted to pH 4.5, methanol and acetonitrile. A photo‐diode array detector set at 245 nm was used for detection. The flow rate was set at 0.3 mL/min. The procedure had good specificity, linearity (0.02–3.14 μg/mL), recovery (96.1–103.9%), limit of detection (0.01–0.02 μg/mL), limit of quantitation (0.03–0.05 μg/mL), and robustness. The correction factors of the process‐related substances were calculated.     

Determination of Sumatriptan and Zolmitriptan in Presence of Their Corresponding Degradation Products by HPTLC Methods

Accurate, sensitive, and precise high performance thin layer chromatographic (HPTLC) methods were developed and validated for the determination of sumatriptan and zolmitriptan in presence of their degradation products. Sumatriptan was separated from its degradation products and analyzed on TLC silica gel 60 F₂₅₄ plates using chloroform–ethyl acetate–methanol–ammonia (4:3:3:0.1, v/v) as a developing system followed by densitometric measurement of the bands at 228 nm. Zolmitriptan was determined using chloroform–ethyl acetate–methanol–ammonia (3:3:3:1, v/v) as a developing system followed by densitometric measurement at 222 nm. The methods were validated over a range of 0.5–4 μg/spot for sumatriptan and 0.5–3 μg/spot for zolmitriptan. The proposed methods were successfully applied for the determination of the studied drugs in bulk powder and in their pharmaceutical formulations.     

Validated HPTLC and HPLC methods for determination of fluorometholone and sodium cromoglycate in presence of their impurities and degradation products; application to kinetic study and on rabbit aqueous humor

Fluorometholone (FLM) and Sodium Cromoglycate (CMG) are co-formulated in ophthalmic preparation and showed marked instability under different conditions. Two specific, sensitive and precise stability-indicating chromatographic methods have been developed and validated for their determination in the presence of their degradation products and FLM impurity. Ten components were efficiently separated by them. The first method was HPTLC-spectrodensitometry, where the separation was achieved using silica gel 60?F₂₅₄ HPTLC plates and developing system of ethyl acetate: methanol (9:1, v/v). The second method was a reversed phase HPLC associated with kinetic study of the degradation process and was successfully applied for determination of the studied compounds in spiked rabbit aqueous humor. The mobile phase was acetonitrile: methanol: 0.05?M potassium dihydrogenphosphate (0.1% trimethylamine); pH 2.5, adjusted with orthophosphoric acid (20: 30: 50, by volume). In both methods, the separated components were detected at 240?nm and system suitability was checked. Good correlation was obtained in the range of 0.10–24.00 and 0.20–48.00?µg band^?1, for FLM and CMG by HPTLC. While for HPLC, the linearity ranges from 0.01–50.00 and 0.05–50.00?µg?mL^?1 for both drugs. The methods were applied in pharmaceutical formulation, where they were compared to the reported method with no significant difference.     

Stability‐indicating chromatographic and chemometric methods for environmentally benign determination of canagliflozin and its major degradation product; A comparative study and greenness assessment

Recently, concepts of sustainable developments, like considering the environmental effect of chemicals used and the amount of hazardous wastes produced, has gained much interest. In this work, a recently approved treatment for type II diabetes mellitus, canagliflozin, was quantified along with its degradation product by two eco‐friendly methods. The first was a specific green HPLC method using a C₁₈ column as a stationary phase and a mobile phase consisting of methanol–water (98:2, v/v) pumped at a flow rate of 1 mL/min with UV detection at 225 nm, and using ibuprofen as an internal standard. The second method was a partial least square chemometric method with the wavelength range 220–320 nm and the data was autoscaled as a preprocessing step for determination of canagliflozin and its degradation product. The greenness profile of the developed methods was studied and compared with the reported methods. The proposed methods were suitable alternatives for the environmentally harmful reported methods for quality control analyses of canagliflozin‐containing samples, analysis of pharmaceutical formulations and sensitive tracing of its possible degradation product. The methods were validated as per International Conference on Harmonization guidelines and statistically compared with the reported HPLC method.     

Study of the degradation behavior of dapagliflozin propanediol monohydrate and metformin hydrochloride by a stability-indicating high-performance thin-layer chromatographic method

A simple, selective, precise, rapid and accurate stability-indicating high-performance thin-layer chromatography (HPTLC) method was developed and validated for the estimation of dapagliflozin and metformin in tablet dosage form. In this work, methanol–ethyl acetate–ammonium acetate (6:4:0.1, V/V) as the mobile phase and aluminum-backed TLC plates pre-coated with 250 µm layer of silica gel 60F₂₅₄ as the stationary phase were used for the estimation of dapagliflozin and metformin. The wavelength selected for detection was 220 nm. The linearity range was found to be 20–100 ng/spot (r² = 0.9985) for dapagliflozin and 500–2500 ng/spot (r² = 0.9984) for metformin. Validation of the developed method was performed as per the International Council for Harmonisation (ICH) guidelines. Stress testing of dapagliflozin and metformin was performed under acidic, alkaline, oxidative, photolytic and dry-heat degradation conditions. The chromatographic conditions successfully resolved dapagliflozin and metformin from their degradation products, formed under various stress conditions. From stress testing, dapagliflozin was found to be significantly degrading under acidic, alkaline, oxidative, photolytic and dry-heat degradation conditions, while metformin was found to be significantly degrading in acidic and alkaline degradation conditions and stable under oxidative, photolytic and dry-heat degradation conditions. Tablet dosage form of dapagliflozin and metformin was analyzed by the developed method.

     

Determination of rifampicin in human plasma by high‐performance liquid chromatography coupled with ultraviolet detection after automatized solid–liquid extraction

A precise and accurate high‐performance liquid chromatography (HPLC) quantification method of rifampicin in human plasma was developed and validated using ultraviolet detection after an automatized solid‐phase extraction. The method was validated with respect to selectivity, extraction recovery, linearity, intra‐ and inter‐day precision, accuracy, lower limit of quantification and stability. Chromatographic separation was performed on a Chromolith RP₈ column using a mixture of 0.05 m acetate buffer pH 5.7–acetonitrile (35:65, v/v) as mobile phase. The compounds were detected at a wavelength of 335 nm with a lower limit of quantification of 0.05 mg/L in human plasma. Retention times for rifampicin and 6,7‐dimethyl‐2,3‐di(2‐pyridyl) quinoxaline used as internal standard were respectively 3.77 and 4.81 min. This robust and exact method was successfully applied in routine for therapeutic drug monitoring in patients treated with rifampicin.