A sensitive LC–MS/MS method for the determination of triptolide and its application to pharmacokinetic research in rats

Triptolide is one of the main active ingredients of Tripterygium wilfordii Hook. F. In this study, a sensitive LC–MS/MS method was established and validated to determine the concentration of triptolide in rat plasma. Triptolide and an internal standard [(5R)‐5‐hydroxytriptolide] were extracted from 100 μL of rat plasma with acetonitrile, and the dried residue was then reconstituted and reacted with benzylamine to produce benzylamine triptolide and benzylamine (5R)‐5‐hydroxytriptolide. Derivatization increased the sensitivity of triptolide detection by ~100‐fold. Quantification was performed using a QTRAP 5500 tandem mass spectrometer with positive electrospray ionization in multiple reaction monitoring mode with an ion transition m/z 468.5 → 192.0 for benzylamine triptolide and m/z 484.3 → 192.1 for benzylamine (5R)‐5‐hydroxytriptolide. Good linearity was observed in the range of 0.030–100 ng/mL with a lower limit of quantitation of 0.030 ng/mL. The intra‐ and inter‐day precision was <6.5%, and the accuracy ranged from ?11.7 to ?4.4%. The recovery remained consistent and was reproducible at different concentrations. This method was successfully applied to the study of triptolide drug–drug interactions in Sprague–Dawley rats. With the use of itraconazole (40 mg/kg, p.o.) as a CYP3A inhibitor, the plasma exposure of triptolide in rats was increased by 36%.     

New catalytic ultrasound method for derivatization of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid in urine, with analysis by GC-MS/MS

A new procedure is described for the derivatization by silylation of 11-nor-Δ⁹-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) present in urine, followed by analysis using gas chromatography–tandem mass spectrometry. A conventional procedure for derivatization of the analyte was evaluated using two types of experimental design. A 2³ factorial design considered the parameters temperature, reaction time, and the solvent/derivatization agent ratio. A central composite design (CCD) was applied to optimize the values of the significant variables. The optimum conditions were a reaction temperature of 50 °C, a reaction time of 30 min, and a BSTFA/acetone ratio of 40:20. The use of imidazole as a catalyst, together with ultrasonication, reduced the reaction time to 5 min and increased the efficiency of derivatization of THCCOOH, compared with the conventional method. The operating conditions of the tandem mass spectrometer were also optimized. The method was linear in the concentration range 1–50 ng mL⁻¹ (R ² = 0.9951). Intra- and inter-day precisions were 7.7–12.3% and 11.1–13.9%, respectively, recoveries ranged between 91 ± 8% and 101 ± 12%, accuracy (as % bias) was between –11.7% and +0.7%, and limits of detection and quantification were 0.5 and 1.0 ng mL⁻¹, respectively.     

Simultaneous Determination of Triptolide and Tripdiolide in Extract of Tripterygium wilfordii Hook. f. by LC–APCI-MS

A novel method using liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry in the negative selected ion monitoring mode has been developed and validated for rapid simultaneous determination of triptolide and tripdiolide in the extract of Tripterygium wilfordii Hook. f. The molecular ions m/z [M–H]⁻ 359 and 375 were selected for the quantification in selected ion monitoring mode for triptolide and tripdiolide. Standard calibration curve was linear over the concentration range of 0.12–24 and 0.15–30 μg mL⁻¹ for triptolide and tripdiolide. The relative standard deviations of intra- and inter-day were in the range of 4.7–9.9 and 8.9–12.6%. The average recoveries were between 96.4 and 104.6%. The limits of quantitation were 2.0 × 10⁻³ and 2.5 × 10⁻³ μg mL⁻¹ for triptolide and tripdiolide.     

Quantitative Analysis of Triptolide in Human Whole Blood by LC–APCI-IT-MS-MS

In this study, the development and validation of an analytical method for triptolide in whole blood using high-performance liquid chromatography coupled with atmospheric-pressure chemical ionization ion trap tandem mass spectrometry (LC–APCI-IT-MS-MS) is reported. This is the first report of the systematic development and validation of an LC–MS-MS method for the quantitation of triptolide in human whole blood using prednisolone as an internal standard (IS). Prior to LC–MS-MS analysis, liquid–liquid extraction with ethyl acetate was used to isolate them from the biological matrix. Validation parameters such as specificity/selectivity, limit of quantitation (LOQ), linearity, precision, accuracy and stability are evaluated for this method. The calibration curve was linear (r ² = 0.9973) in the concentration range of 0.5–100.0 ng mL⁻¹ in human whole blood with a lower limit of quantitation of 0.5 ng mL⁻¹. Intra- and inter-day relative standard deviations (RSDs) were less than 8.6 and 11.7%, respectively. Extraction recoveries of triptolide ranged from 81.5 to 88.1%. This assay can be used to determine trace triptolide in human whole blood.     

Chiral Separation of (R,R)-Tadalafil and Its Enantiomer in Bulk Drug Samples and Pharmaceutical Dosage Forms by Chiral RP-LC

A new simple isocratic chiral RP-LC method has been developed for the separation and quantification of the enantiomer of (R,R)-tadalafil in bulk drugs and dosage forms with an elution time of about 20 min. Chromatographic separation of (R,R)-tadalafil and its enantiomer was achieved on a bonded macro cyclic glycopeptide stationary phase. The method resolves the (R,R)-tadalafil and its enantiomer with a resolution (R _s) greater than 2.4 in the developed chiral RP-LC. The mobile phase used for the separation and quantification of (R,R)-tadalafil and its enantiomer involves a simple mixture of reverse phase solvents and the cost of analysis was drastically decreased. The test concentration is 0.4 mg mL⁻¹ in the mobile phase. This method is capable of detecting the enantiomer of (R,R)-tadalafil up to 0.0048 μg wrt test concentration 400 μg mL⁻¹ for a 20 μL injection volume. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. There was no interference of degradants with (R,R)-tadalafil and its enantiomer in the developed method. The developed chiral RP-LC method was validated with respect to linearity, accuracy, precision and robustness. The percentage recovery for the enantiomer of (R,R)-tadalafil in bulk drug samples and in dosage forms ranged from 97.0 to 102.5%. The test solution was found to be stable in the mobile phase for 48 h after preparation.     

In situ derivatization hollow fibre liquid-phase microextraction for the determination of biogenic amines in food samples

Hollow fibre liquid-phase microextraction with in situ derivatization using dansyl chloride has been successfully developed for the high-performance liquid chromatography-ultraviolet (HPLC-UV) determination of the biogenic amines (tryptamine, putrescine, cadaverine, histamine, tyramine, spermidine) in food samples. Parameters affecting the performance of the in situ derivatization process such as type of extraction solvent, temperature, extraction time, stirring speed and salt addition were studied and optimized. Under the optimized conditions (extraction solvent, dihexyl ether; acceptor phase, 0.1 M HCl; extraction time, 30 min; extraction temperature, 26 °C; without addition of salt), enrichment factors varying from 47 to 456 were achieved. Good linearity of the analytes was obtained over a concentration range of 0.1–5 μg mL⁻¹ (with correlation coefficients of 0.9901–0.9974). The limits of detection and quantification based on a signal-to-noise ratio of 3–10, ranged from 0.0075 to 0.030 μg mL⁻¹ and 0.03 to 0.10 μg mL⁻¹, respectively. The relative standard deviations based on the peak areas for six replicate analysis of water spiked with 0.5 μg mL⁻¹ of each biogenic amine were lower than 7.5%. The method was successfully applied to shrimp sauce and tomato ketchup samples, offering an interesting alternative to liquid–liquid extraction and solid phase extraction for the analysis of biogenic amines in food samples.     

N-alkylpyridinium quaternization combined with liquid chromatography–electrospray ionization-tandem mass spectrometry: A highly sensitive method to quantify fatty alcohols in thyroid tissues

A highly sensitive method was developed for the identification and quantification of fatty alcohols in biological tissues. In the presence of pyridine-d₀ and triflic anhydride (Tf₂O), fatty alcohols were converted into permanently charged N-alkylpyridinium ions. Stable isotope-labeled derivatives were generated by pyridine-d₅ and added as internal standard (IS). The mixture was analyzed by liquid chromatography coupled to positive electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS). This method was optimized and validated in terms of reaction time, derivatization efficiency, stability, desalting, and ion suppression effect. Besides, fatty alcohols exhibited good linear relationship (r² > 0.993) over the concentration range of 10 ng mL⁻¹–1 μg mL⁻¹. The limits of detection (LODs) were lowered from previously reported 0.1 ng mL⁻¹ to 0.25 pg mL⁻¹. Precision (RSD% < 15.6%), accuracy (93.0–107.2%), matrix effect, and recovery (in thyroid tissues) were validated as well. Finally, this method was applied for the analysis of ten even carbon-numbered fatty alcohols (C₈–C₂₄) in human thyroid carcinoma and para-carcinoma tissues, revealing a significant decrease of fatty alcohols (free and esterified) in thyroid carcinoma tissues (p < 0.05).     

Study of the metabolism on tobacco-specific N-nitrosamines in the rabbit by solid-phase extraction and liquid chromatography–tandem mass spectrometry

Metabolism of four tobacco-specific N-nitrosamines (TSNAs), N′-nitrosonornicotine (NNN), N′-nitrosoanatabine (NAT), N′-nitrosoanabasine (NAB), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) has been studied by solid-phase extraction (SPE) and liquid chromatography–tandem mass spectrometry (LC–MS–MS). 4-(Methylnitrosamino)-4-(3-pyridyl)-1-butanol (iso-NNAL) was used as internal standard. SPE and LC–MS–MS was found to be a rapid, simple, sensitive, and selective method for analysis of TSNAs in rabbit serum. The relative standard deviation (R.S.D., n = 6) for analysis of 5 ng mL⁻¹ and 0.5 ng mL⁻¹ standards and of serum sample spiked with 5 ng mL⁻¹ standards of five TSNAs was 2.1–11% and recovery of 5 ng mL⁻¹ standards from serum was 100.2–112.9%. A good linear relationship was obtained between peak area ratio and concentration in the range of 0.2–100 ng mL⁻¹ for NNAL and 0.5–100 ng mL⁻¹ for other four TSNAs, with correlation coefficients (R ²) >0.99 (both linear and log–log regression). Detection limits for standards in solvent were between 0.04 and 0.10 ng mL⁻¹. Doses of TSNAs administered to rabbits via the auricular vein were 4.67 μg kg⁻¹ and 11.67 μg kg⁻¹, in accordance with the different levels in cigarettes. Metabolic curves were obtained for the four TSNAs and for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a metabolite of NNK; on the basis of these curves we modeled metabolic kinetic equations for these TSNAs by nonlinear curve fitting.     

LC-MS-MS Determination of (S,R)-Penehyclidine in Rat Plasma

A sensitive and selective method for determination of (S,R)-penehyclidine in rat plasma by liquid chromatography-tandem mass spectrometry is described. The procedure employed the use of an internal standard (I.S.) and a simple protein precipitation step. The method developed was linear from 0.1 to 100 ng mL⁻¹, with a sensitivity of 0.1 ng mL⁻¹ as the lower limit of quantification. The intra- and inter-day assay accuracy (relative error) was within 8.27% and precision (RSD) was below 6.7%. It was successfully applied to pharmacokinetic studies of (S,R)-penehyclidine in rat plasma.

     

Automated high-performance liquid chromatographic determination of amphetamine in biological fluids using column-switching and on-column derivatization

Summary A rapid and simple liquid-chromatographic method has been developed for on-line quantification of amphetamine in biological fluids. Untreated samples (20 μL) are injected directly into the chromatographic system and purified on a 20 mm×2.1 mm i.d. pre-column packed with 30 μm Hypersil C₁₈ stationary phase. After clean-up the analyte is transferred to the analytical column (125 mm×4 mm i.d., 5 μm LiChrospher 100 RP₁₈) for derivatization and separation using a mixture of acetonitrile and the derivatization reagent (o-phthaldialdehyde andN-acetyl-L-cysteine) as the mobile phase. The experimental conditions for on-line derivatization and resolution of the amphetamine have been optimized, and the results have been compared with those obtained by derivatizing the analyte in pre-column mode. The method described has been applied to the determination of amphetamine in plasma and urine. Good linearity and reproducibility were obtained in the 0.1–10.0 μg mL⁻¹ concentration range, and limits of detection were 25 ng mL⁻¹ and 10 ng mL⁻¹ with UV and fluorescence detection, respectively. The procedure described is very simple and rapid, because no off-line manipulation of the sample is required; the total analysis time is approximately 8 min.     

Determination of perfluorocarboxylic acids in water by ion-pair dispersive liquid–liquid microextraction and gas chromatography–tandem mass spectrometry with injection port derivatization

A novel technique for derivatization in a gas chromatograph injection port after a one-step extraction of trace perfluorocarboxylic acids (PFCAs) in water with ion pair formation during dispersive liquid–liquid microextraction (DLLME) was investigated. Tetrabutylammonium hydrogen sulfate (TBAHS) was used as the ion pair reagent. PFCA butyl ester derivatives were formed in the GC injection port and then analyzed using gas chromatography coupled to tandem mass spectrometry with negative chemical ionization. According to our analysis, the operative linear range for PFCA detection from 250 pg mL⁻¹ to 2 μg mL⁻¹ with a relative standard derivation (RSD) below 13%. Detection limits were achieved at the level of 37–51 pg mL⁻¹. This method was successfully applied for the analyzing of PFCAs in river water samples from urban and industrial areas without tedious pretreatment. The concentration range over which PFCAs were detected is from 0.6 ng mL⁻¹ to 604.9 ng mL⁻¹.     

Determination of Apigenin by LC with Electrochemical Detection

A rapid, sensitive, and specific method for quantification of olmesartan, the prodrug of olmesartan medoxomil, in human plasma, using zidovudine as internal standard, is described. Sample preparation involved a simple solid-phase extraction procedure. The extract was analyzed by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry (LC–MS–MS). Chromatography was performed isocratically on a 5 μm C₁₈ analytical column (50 mm × 4.6 mm i.d.) with water–acetonitrile–formic acid 20:80:0.1 (v/v) as mobile phase. The response to olmesartan was a linear function of concentration over the range 4.82–1,928 ng mL⁻¹. The lower limit of quantification in plasma was 4.82 ng mL⁻¹. The method was successfully applied in a bioequivalence study of an olmesartan formulation after administration as a single oral dose.     

Bulk derivatization and cation exchange restricted access media-based trap-and-elute liquid chromatography–mass spectrometry method for determination of trace estrogens in serum

Estrone (E1), estradiols (α/β-E2), and estriol (E3) are four major metabolically active estrogens exerting strong biological activities at very low circulating concentrations. This paper reports a sensitive and efficient method with automated, on-line clean-up and detection to determine trace estrogens in a small volume of serum samples using liquid chromatography–electrospray ionization–tandem mass spectrometry directly, without off-line liquid–liquid or solid-phase extraction pretreatments. Serum aliquots (charcoal stripped fetal bovine serum, 100 μL) were spiked with four estrogen standards and their corresponding isotope-labeled internal standards, then bulk derivatized with 2-fluoro-1-methyl-pyridium p-toluenesulfonate (2-FMP) to establish the calibration curves and perform method validation. Calibration was established in the concentration ranges of 5–1000 pg mL⁻¹, and demonstrated good linearity of R² from 0.9944 to 0.9997 for the four derivatized estrogens. The lower detection limits obtained were 3–7 pg mL⁻¹. Good accuracy and precision in the range of 86–112% and 2.3–11.9%, respectively, were observed for the quality control (QC) samples at low, medium, and high concentration levels. The stability tests showed that the derivatized serum samples were stable 8 h after derivatization at room temperature and at least to 48 h if stored at −20 °C. The method was applied to measure trace estrogens in real human and bovine serum samples, and three of four estrogen compounds studied were observed and quantified.     

Simple derivatization of aldehydes with d-cysteine and their determination in beverages by liquid chromatography–tandem mass spectrometry

We describe a simple derivatization method to determine aldehydes. This method is based on derivatization with d-cysteine and consecutive liquid chromatography–tandem mass spectrometry (LC–MS/MS). The optimum derivatization conditions of aldehydes with d-cysteine were 10 min at 50 °C and pH 7.0. The formed alkyl thiazolidine-4-carboxylic acid derivatives were directly injected in LC–MS/MS. In the established condition, the method was used to detect eight aldehydes in beverages. The limit of detection (LOD) and limit of quantification (LOQ) of the aldehydes were 0.2–1.9 μg L⁻¹ and 0.7–6.0 μg L⁻¹ and the relative standard deviation was less than 2.0% at concentrations of 0.1 mg L⁻¹ and 1.0 mg L⁻¹ with the exception of octanal. All the beverage samples had detectable levels of methanal (0.033–0.145 mg L⁻¹), ethanal (0.085–2.12 mg L⁻¹), propanal (ND to 0.250 mg L⁻¹), butanal (ND to 0.003 mg L⁻¹), pentanal (ND to 0.471 mg L⁻¹), hexanal (ND to 0.805 mg L⁻¹), heptanal (0.019–3.91 mg L⁻¹) and octanal (0.029–0.118 mg L⁻¹).     

A Validated Chiral LC Method for the Enantiomeric Separation of Repaglinide on Amylose Based Stationary Phase

A simple, isocratic, normal phase chiral HPLC method was developed and validated for the enantiomeric separation of repaglinide, (S)-(+)-2-ethoxy-4-N [1-(2-piperidinophenyl)-3-methyl-1-butyl] aminocarbonylmethyl] benzoic acid, an antidiabetic in bulk drug substance. The enantiomers of repaglinide were resolved on a ChiralPak AD-H (amylose based stationary phase) column using a mobile phase consisting of n-hexane: 2-propanol:trifluoroacetic acid (95:5:0.2 v/v/v) at a flow rate of 1.0 mL min⁻¹. The resolution between the enantiomers was found to be not >3.5 in optimized method. The presence of trifluoroacetic acid in the mobile phase played an important role, in enhancing chromatographic efficiency and resolution between the enantiomers. The developed method was extensively validated and proved to be robust. The calibration curve for (R)-enantiomer showed excellent linearity over the concentration range of 900 ng mL⁻¹ (LOQ) to 6,000 ng mL⁻¹. The limit of detection and limit of quantification for (R)-enantiomer were 300 and 900 ng mL⁻¹, respectively. The percentage recovery of the (R)-enantiomer ranged between 98.3 and 101.05% in bulk drug samples of repaglinide. Repaglinide sample solution and mobile phase were found to be stable up to 48 h. The developed method was found to be enantioselective, accurate, precise and suitable for quantitative determination of (R)-enantiomer in bulk drug substance.     

A fast derivatization procedure for gas chromatographic analysis of perfluorinated organic acids

A rapid and simple derivatization procedure has been developed for gas chromatographic determination of perfluorinated organic acids (PFCAs, C₆–C₁₂), using isobutyl chloroformate (IBCF) to convert the acids into the more volatile isobutyl esters, under catalysis by pyridine. The procedure was optimized in an acetonitrile medium and applied to GC techniques with electron-capture detection (GC-ECD) and mass spectrometry with electron-impact ionization (GC-EI-MS); for the sake of comparison, HPLC with electrospray-ionization MS (HPLC-ESI(−)-MS) was also tested. The LOD and LOQ values obtained for these three techniques were compared, and the lowest LODs were obtained with GC-ECD (0.06–1.80 μg mL⁻¹). The procedure was further optimized in an aqueous medium, obtaining the best results in a phosphate buffer (pH 2.5, 50 mmol L⁻¹), in which the LOD and LOQ values were measured for GC-ECD a GC-EI-MS. The lowest LODs were found for GC-EI-MS (0.030–0.314 μg mL⁻¹). The practical applicability was tested on Vltava river water samples.     

A chitosan–polypyrrole magnetic nanocomposite as μ-sorbent for isolation of naproxen

An extracting medium based on chitosan–polypyrrole (CS–PPy) magnetic nanocomposite was synthesized by chemical polymerization of pyrrole at the presence of chitosan magnetic nanoparticles (CS-MNPs) for micro-solid phase extraction. In this work, magnetic nanoparticles, the modified CS-MNPs and different types of CS–PPy magnetic nanocomposites were synthesized. Extraction efficiency of the CS–PPy magnetic nanocomposite was compared with the CS-MNPs and Fe₃O₄ nanoparticles for the determination of naproxen in aqueous samples, via quantification by spectrofluorimetry. The scanning electron microscopy images obtained from all the prepared nanocomposites revealed that the CS–PPy magnetic nanocomposite possess more porous structure. Among different synthesized magnetic nanocomposites, CS–PPy magnetic nanocomposite showed a prominent efficiency. Influencing parameters on the morphology of CS–PPy magnetic nanocomposite such as weight ratio of components was also assayed. In addition, effects of different parameters influencing the extraction efficiency of naproxen including desorption solvent, desorption time, amount of sorbent, ionic strength, sample pH and extraction time were investigated and optimized. Under the optimum condition, a linear calibration curve in the range of 0.04–10 μg mL⁻¹ (R² = 0.9996) was obtained. The limits of detection (3S_b) and limits of quantification (10S_b) of the method were 0.015 and 0.04 μg mL⁻¹ (n = 3), respectively. The relative standard deviation for water sample spiked with 0.1 μg mL⁻¹ of naproxen was 3% (n = 5) and the absolute recovery was 92%. The applicability of method was extended to the determination of naproxen in tap water, human urine and plasma samples. The relative recovery percentages for these samples were in the range of 56–99%.     

Determination and Pharmacokinetic Study of 10-O-Dimethylaminoethylginkgolide B in Rat Plasma by LC–MS

To evaluate the pharmacokinetics of a novel analogue of ginkgolide B, 10-O-dimethylaminoethylginkgolide B (XQ-1) in rat plasma in pre-clinical studies, a sensitive and specific liquid chromatographic method with electrospray ionization mass spectrometry detection (LC–ESI–MS) was developed and validated. After a simple extraction with ethyl acetate, XQ-1 was analyzed on a Shim-pack C18 column with a mobile phase of a mixture of 1 μmol L⁻¹ ammonium acetate containing 0.02% formic acid and methanol (55:45, v/v) at a flowrate of 0.3 mL min⁻¹. Detection was performed in selected ion monitoring (SIM) mode using target ions at [M + H]⁺ m/z 496.05 for XQ-1 and m/z 432.10 for the internal standard (lafutidine). Linearity was established for the concentration range from 2 to 1,000 ng mL⁻¹ . The extraction recoveries ranged from 86.0 to 89.9% in plasma at concentrations of 5, 50, and 500 ng mL⁻¹. The lower limit of quantification was 2 ng mL⁻¹ with 100 μL plasma. The validated method was successfully applied to a pharmacokinetic study after intragastic administration of XQ-1 mesylate in rats at a dose of 20 mg kg⁻¹.     

Rapid and sensitive gas chromatography–ion-trap tandem mass spectrometry method for the determination of tobacco-specific N-nitrosamines in secondhand smoke

Tobacco-specific nitrosamines (TSNAs) are some of the most potent carcinogens in tobacco and cigarette smoke. Accurate quantification of these chemicals is needed to help assess public health risks. We developed and validated a specific and sensitive method to measure four TSNAs adsorbed to model surfaces and secondhand smoke (SHS) particles using gas chromatography–ion-trap tandem mass spectrometry. In an 18-m³ room-sized chamber, a smoking machine generated realistic concentrations of SHS that were actively sampled on Teflon-coated fiber glass (TCFG) filters, and passively sampled on cellulose substrates. A simple solid–liquid extraction protocol using methanol as solvent was successfully applied to both substrates with recoveries ranging from 85 to 115%. For each TSNA, tandem MS parameters were optimized and the major fragmentation pathways were elucidated. The method showed excellent performance, with a linear dynamic range from 2 to 1000 ng mL⁻¹, low detection limits (S/N > 3) of 30–300 pg mL⁻¹ and precision with experimental errors below 10% for all compounds. Moreover, no interfering peaks were observed, indicating a high selectivity of MS/MS without the need for a sample clean-up step. This method provides a suitable analytical tool to detect and quantify traces of TSNA in indoor environments polluted with SHS.     

Determination and Validation of an LC Method for Fluvoxamine in Tablets

A new, simple, rapid and specific reversed-phase high-performance liquid chromatography (HPLC) method was developed and validated for the determination of fluvoxamine in pharmaceutical dosage forms. The HPLC separation was achieved on a C₁₈ μ-Bondapack column (250 mm × 4.6 mm) using a mobile phase of acetonitrile–water (80:20, v/v) at a flow rate of 1 mL min⁻¹. Proposed method is based on the derivatization of fluvoxamine with 1,2-naphthoquinone-4-sulphonic acid sodium salt (NQS) in borate buffer of pH 8.5 to yield a orange product. The HPLC method is based on measurement of the derivatized product using UV-visible absorbance detection at 450 nm. The method was validated for specificity, linearity, precision, accuracy, robustness. The degree of linearity of the calibration curves, the percent recoveries of fluvoxamine, the limit of detection and quantification, for the HPLC method were determined. The assay was linear over the concentration range of 45–145 ng mL⁻¹ (r = 0.9999). Limit of detection and quantification for fluvoxamine were 15 and 50 ng mL⁻¹, respectively. The results of the developed procedure (proposed method) for fluvoxamine content in tablets were compared with those by the official method. The method was found to be simple, specific, precise, accurate, reproducible and robust.