XTerra C18 Column Research Articles

A simple and rapid external standard calibration HPLC method for determination of lumefantrine in dried blood spot samples from malaria patients in Botswana.

A simple external calibration liquid chromatography-diode array detector method was developed, validated, and applied for the determination of lumefantrine (Lum) in dried blood spot (DBS) samples collected from malaria patients in Botswana. The samples were validated in accordance with the United States Food and Drug Administration guidelines for bioanalytical methods after sample preparation using solid-liquid extraction. Separation was achieved using an XTerra C18 column (50 × 4.6mm,5μm), and a binary solvent system of acetonitrile and water adjusted to pH2.3 was used as the mobile phase. The validated method was applied for the determination of Lum in DBS samples collected from malaria patients infected with Plasmodium falciparum in Botswana. The calibration curve was linear between 0.5 and 12 μg/mL with a coefficient of determination (R2 ) of 0.9996. The limit of detection and the lower limit of quantification were 0.5 and 1.4μg/mL, respectively. The efficiency of extraction measured as percentage recovery ranged between 84.2% and 107.8% at the three quality control (QC) levels, that is, low QC, mid QC, and high QC. In conclusion, data suggest that the method is suitable for the determination of trace Lum in biofluids and can also be used for therapeutic drug monitoring and pharmacokinetic profiling.

Development and validation of HPLC method for simultaneous determination of amlodipine besylate, telmisartan and hydrochlorothiazide in combined tablets

A combination of three active ingredients amlodipine besylate, telmisartan and hydrochlorothiazide has been used and proven effective in the treatment of hypertension in patients with moderate to severe hypertension, while it contributes to a reduction in product costs and increases patient compliance. However, there are no monographs in pharmacopoeias for the simultaneous quantification of these three active ingredients. Therefore, in order to contribute to the establishment of an in-house specification for domestic generic drugs, shorten the time of quantifying each ingredient separately, the study was carried out with the aim of developing and validating procedure for the simultaneous quantification of amlodipine besylate, telmisartan and hydrochlorothiazide by high performance liquid chromatography. In this study, we conducted investigation into different chromatographic conditions, developed an analytical procedure for the simultaneous quantification of amlodipine besylate, telmisartan and hydrochlorothiazide in tablets preparation. The procedure was validated in compliance with ICH guidelines. Suitable chromatographic conditions for simultaneous quantification of amlodipine besylate, telmisartan and hydrochlorothiazide are: Xterra C18 column (250 x 4.6 mm, 5 μm), detection wavelength 230 nm, gradient program of mobile phase: acetonitrile and phosphate buffer pH 3.0, flow rate 1.0 ml/min, sample injection volume 10 μl, column temperature 30°C. The procedure was validated for systematic suitability, accuracy, specificity, linearity, precision, working range and robustness. A procedure for the simultaneous quantification of amlodipine besylate, telmisartan and hydrochlorothiazide in tablets by high performance liquid chromatography has been successfully developed and validated.

Sustainable liquid chromatographic determination and purity assessment of a possible add-on triple-action over-the-counter pharmaceutical combination in COVID-19

Nowadays, all researchers are focused on combating the pandemic COVID-19. According to recent statistics, most patients are managed at home. An over-the-counter (OTC) triple action formula containing paracetamol (PAR), aspirin (ASP), and diphenhydramine (DIPH) is widely prescribed for pain, fever and as night-time sleep aid. For COVID-19 patients, this combination is now suggested as part of symptomatic therapy and prophylaxis. In this work, two simple liquid chromatographic approaches were designed for simultaneous determination of PAR, ASP, and DIPH in Excedrin® PM caplets, beside three specified official toxic impurities, namely, p-aminophenol, p-nitrophenol, and salicylic acid. The first method comprised high-performance thin-layer chromatographic separation coupled with densitometric quantification, on silica gel HPTLC 60 F254 aluminium sheets as the stationary phase, ethyl acetate–methanol-aqueous ammonium hydroxide (10.0: 2.0: 0.1, by volume) as the developing system and scanning was performed at 210.0 nm. The second one is a high-performance liquid chromatography coupled with diode array detector. Successful separation of the six components was performed on XTerra C18 column with isocratic elution of mobile phase 0.1% triethylamine acidified water: methanol (70:30, v/v) adjusted with o-phosphoric acid to pH 3.0 and methanol (90:10, v/v) with flow rate programming and detection at 210.0 nm. Validation of the proposed methods was performed according to ICH guidelines. Both methods were successfully used for quality control of the cited drugs in their marketed formulation. Moreover, the in-vitro release study was monitored using the proposed HPLC-DAD method. The greenness profile of the proposed methods was assessed and comparatively evaluated through various assessment tools, specifically; the analytical eco-scale system, national environmental method index (NEMI), green analytical procedure index (GAPI) and analytical greenness (AGREE) metric.

Simultaneous Quantitation of Paracetamol and Lornoxicam in the Presence of Five Related Substances and Toxic Impurities by a Selective HPLC-DAD Method.

This research describes the simultaneous quantitation of paracetamol (PRM) and lornoxicam (LRX) with five of their related substances and toxic impurities, including, 4-nitrophenol (NTP), 4-aminophenol (AMP), 4-chloroacetanilide (CAC), N-phenylacetamide (NPA), and 2-aminopyridine (APD) using a specific HPLC-diode array detector (DAD) method. The chromatographic separation involves the use of a XTerra C18 column as the stationary phase and a mobile phase consisting of acetonitrile and 0.025 M phosphate buffer (pH 6). The separation was performed using gradient elution mode at 1.0 mL/min flow rate and detection at 260 nm for the determination of PRM and LRX. For detecting PRM and LRX in the presence of their toxic impurities, 270 nm was used. Validation of the suggested HPLC method was accomplished with regard to linearity, ranges, detection and quantitation limits, robustness, accuracy, precision, and specificity. Excellent resolution of the mixture components was accomplished at retention times 4.2, 4.8, 7.4, 11.1, 13.5, 14.7, and 15.3 min for APD, AMP, PRM, NPA, LRX, NTP, and CAC, respectively. Linearity was established for PRM and LRX within concentration ranges of 10-100 and 10-60 µg/mL, respectively. The correlation coefficients obtained were >0.9997. The suggested method was confirmed to be a specific stability-indicating through the selective separation of PRM and LRX from their related substances, degradants, and impurities. The proposed method was successfully utilized for the sensitive and selective determination of PRM and LRX in their pharmaceutical formulation. To the best of our knowledge, this is the first impurity profiling assay method for this combination in the presence of five of their toxic related substances and impurities. Taking into consideration that at least two of the studied impurities (AMP and APD) are actually reported degradation products for the main drugs, the suggested method can be considered stability-indicating as well.

A Novel Stability Indicating RP-HPLC Method for Simultaneous Quantification of Serdexmethylphenidate and Dexmethylphenidate in Fixed Dosage Form.

A simple, robust stability indicating RP-HPLC method was developed for simultaneous quantification of serdexmethyl phenidate and dexmethyl phenidate in a fixed capsule dosage form. This is the first method to be reported for simultaneous estimation and quantification of degradation products produced from forced stressing of the dosage form as per ICH guidelines. The chromatographic separation was attained on Waters X-terra C18 column using a mixture of trifluoro acetic acid and acetonitrile (70:30v/v) as mobile phase with a flow rate of 1mL, monitored at 265nm over a run time of 10min. Serdexmethyl phenidate and dexmethyl phenidate were eluted with retention times of 2.71min and 7.33min, respectively. The method displayed linear responses in the range of 4.2-63μg/mL (0.9994) for serdexmethyl phenidate and 0.9 to 3.5μg/mL (0.9998) for dexmethyl phenidate, respectively. The percentage recoveries of the two drugs were found within the acceptable limits. Forced degradation was conducted and showed considerable degradation in various stress conditions. It also confirms the specificity of the method as no interference peaks were observed concerning for to stress products. This method can be routinely used in quality control labs for simultaneous determination of respective drugs in marketed dosage form.

BIOANALYTICAL RP-HPLC METHOD DEVELOPMENT AND VALIDATION OF CLOPIDOGREL BISULFATE IN WISTAR RAT PLASMA AND ITS APPLICATION TO PHARMACOKINETIC STUDY

Objective: A novel, simple, precise, accurate, sensitive, and reproducible HPLC method for determining clopidogrel bisulfate in Wistar rat plasma was developed and validated. Methods: The chromatographic separation was performed using Xterra C18 (250 x 4.6 mm, 5μ) column. Mobile phase composed of Acetonitrile ACN: 0.05M potassium dihydrogen orthophosphate buffer pH 4.2 and in the ratio of 75:25% v/v at a flow rate of 1.2 ml/min. Detection was carried out using a PDA detector at 220 nm. The bioanalytical clopidogrel method was validated as per ICH guidelines. Results: The selected chromatographic condition was found to efficiently separate clopidogrel bisulfate (RT-2.838 min). The calibration curve was linear over the concentration range 40-200 ng/ml in Wistar rat plasma with a correlation coefficient of 0.999, respectively. The precision study revealed that the cumulative percentage variation was within the acceptable limit, and accuracy research showed the value of mean percent recovery between 99.72-99.83 %. Conclusion: A simple, rapid, specific, accurate, and precise analytical method was developed and validated using Wistar rat plasma. The technique was strictly validated according to the ICH guidelines. Acquired results demonstrate that the proposed strategy can be effortlessly and advantageously applied for routine analysis of clopidogrel in the Wistar rat plasma.

HPLC-DAD approach for determination of pyridoxine HCl and doxylamine succinate in pure and pharmaceutical dosage forms: A green stability-indicating assay method

The environmental impact of analytical procedures is a major concern nowadays. As a result, scientists are now working to reduce the adverseenvironmental impact of chemicals and solvents. Stability testing is an important stage in the pharmaceutical industry because it determines how a drug product's quality changes over time as a result of environmental and laboratory conditions. This study demonstrates the use of green reversed phase (RP-HPLC) to determine pyridoxine HCl (PYR) and doxylamine succinate (DOX) in the presence of DOX oxidative degradation product (DOX DEG). DOX DEG was identified by using (LC-MS). The chromatographic separation was achieved using a mobile phase consisting of ethanol and 0.01 M phosphate buffer pH = 5.0 (10: 90, v/v) with isocratic elution, pumped at 1.0 mL/min on an Xterra C18 column (100 mm × 4.6 mm × 5 m). DAD was used for detection at 254.0 nm and the retention times for PYR, DOX and DOX DEG were 1.41 min, 5.89 min, and 7.36 min, respectively. This procedure was validover linearity ranges of 10.00–120.00 μg/mL and 5.00–100.00 μg/mL, with mean percentage recoveries100.09 ± 0.949 and 100.29 ± 0.519 for PYR and DOX, respectively. The presented chromatographic method was fully validated in accordance with ICH requirements and was statistically compared with those of the reported method and there was no significant difference between them. The proposed approach was shown to be more eco -friendly in terms of the use of hazardous chemicals and solvents, energy consumption, and waste generation than the reported one.

STABILITY INDICATING RP-HPLC METHOD FOR SEPARATION AND QUANTIFICATION OF RELATED SUBSTANCES OF TICLOPIDINE IN BULK AND PHARMACEUTICAL FORMULATIONS

A simple, specific, accurate and stable reverse phase liquid chromatographic method was developed for the simultaneous determination of ticlopidine and its related impurities A and B in bulk drug and tablet dosage forms. The analysis has been performed on XTerra C18 column (250 mm×4.6 mm; 5 µ id) and mobile phase containing of methanol and pH 6.8 phosphate buffer in the ratio of 80:20 (V/V). The detection was carried at 228 nm with a flow rate of 1.0 mL/min. The retention times were found to be 8.9, 5.98 and 4.62 min for ticlopidine, impurities A and B, respectively. The method was validated according to ICH guidelines. The method was validated for specificity, precision, linearity, accuracy and robustness. The linearity range of 50-200 µg/mL for ticlopidine and 0.5-2.0 µg/mL for impurity A and B. The recoveries of ticlopidine and impurities were found to be within the range of 98-102 and the % RSD in each spiked level was found to be less than 2. The stress degradation studies confirmed that the method was effectively separate the degradation products and impurities formed in the stress studies and hence the method was found to be stability indicating method. The method can effectively quantify the standard drug ticlopidine and its impurities A and B in bulk drug and pharmaceutical formulations.

Multivariate optimization and evaluation of quaternary mixture in bulk and co-formulated dosage forms by central composite design

BackgroundThe current study describes the use of central composite design for multivariate optimization of resolution and retention time, taking into account different critical method parameters like organic phase, pH, flow rate, and wavelength for risk assessment. The chromatographic method for the assay of the most effective anti-viral regimen (EPCLUSA, DARVONI, and HARVONI) was developed. An experimental design was presented by sequential investigation of four independent parameters. The method was developed using XTERRA C18 (250 mm × 4.6 mm, 5 μm particle size) column in isocratic mode using potassium dihydrogen phosphate buffer (pH adjusted to 5) and acetonitrile (50:50 % v/v) as mobile phase at a flow rate of 1.0 ml/min and UV detection wavelength of 260 nm.ResultsThe separation of four drugs with fine resolution and preferable retention times was achieved. Retention times of four drugs were found to be 2.96, 3.91, 7.15, and 11.94 min for daclatasvir, sofosbuvir, velpatasvir, and ledipasvir, respectively. The percentage accuracy of labelled claim was in the range of 99–102%, and the pooled %RSD for repeatability, precision, and accuracy was less than 2%.ConclusionThe suggested method was applied for quantification and identification of studied drugs in tablets; the results agreed with the label claim and were validated according to the ICH guidelines. The optimized method can be used for pharmacokinetic and quality control studies.

A new stability-indicating RP-HPLC method for the determination of dicyclomine hydrochloride and dimethicone combination in tablet dosage forms

BackgroundWe describe a “stability-indicating liquid chromatography” technique for the estimation of dimethicone (DEC) and dicyclomine hydrochloride (DEH) in the established tablet formulations. Individual quantification of DEH and DEC was reported. But simultaneous quantification of DEH and DEC was lacking. DEH and DEC were analysed on an “XTerra C18 column (250 mm × 4.6 mm, 5 μm)” with the mobile phase solvent run isocratically with 0.1M K2HPO4-acetonitrile (55:45, v/v) on a flow speed of 1.0 mL/min.ResultsThe chromatographic run period for the DEC and DEH assay was 6.0 min with retention times of 2.134 and 2.865 min, respectively. The method was validated for accuracy (99.453 to 100.417% and 99.703 to 100.303% recovery values for DEH and DEC, respectively), precision (RSV value 0.135% for DEC and 0.171% for DEH), linearity (5–15 μg/mL for DEH and 20–60 μg/mL for DEC), selectivity (no hinderance from excipients) and specificity (no hinderance from degradants) recovery.ConclusionThe developed stability-indicating liquid chromatography process was well applied to established tablet formulations.

Rp-HPLC Determination of Quercetin in a Novel D-α-Tocopherol Polyethylene Glycol 1000 Succinate Based SNEDDS Formulation: Pharmacokinetics in Rat Plasma.

Despite its proven efficacy in diverse metabolic disorders, quercetin (QU) for clinical use is still limited because of its low bioavailability. D-α-Tocopherol polyethylene glycol 1000 succinate (TPGS) is approved as a safe pharmaceutical adjuvant with marked antioxidant and anti-inflammatory activities. In the current study, several QU-loaded self-nanoemulsifying drug delivery systems (SNEDDS) were investigated to improve QU bioavailability. A reversed phase high performance liquid chromatography (RP-HPLC) method was developed, for the first time, as a simple and sensitive technique for pharmacokinetic studies of QU in the presence of TPGS SNEDDS formula in rat plasma. The analyses were performed on a Xterra C18 column (4.6 × 100 mm, 5 µm) and UV detection at 280 nm. The analytes were separated by a gradient system of methanol and phosphate buffer of pH 3. The developed RP-HPLC method showed low limit of detection (LODs) of 7.65 and 22.09 ng/mL and LOQs of 23.19 and 66.96 ng/mL for QU and TPGS, respectively, which allowed their determination in real rat plasma samples. The method was linear over a wide range, (30–10,000) and (100–10,000) ng/mL for QU and TPGS, respectively. The selected SNEDDS formula, containing 50% w/w TPGS, 30% polyethylene glycol 200 (PEG 200), and 20% w/w pumpkin seed oil (PSO), showed a globule size of 320 nm and −28.6 mV zeta potential. Results of the pharmacokinetic studies showed 149.8% improvement in bioavailability of QU in SNEDDS relative to its suspension. The developed HPLC method proved to be simple and sensitive for QU and TPGS simultaneous determination in rat plasma after oral administration of the new SNEDDS formula.

Novel Stability Indicating RP-HPLC Method Development and Validation for Simultaneous Estimation of Alfuzosin and Dutasteride in Pharmaceutical Dosage Form

Abstract: Aim: The present study was to develop a simple, accurate and stable reverse phase liquid chromatographic method and validate in bulk drug and pharmaceutical dosage form for the simultaneous determination of Alfuzosin Hydrochloride (ALF) and Dutasteride (DUT). Materials and Methods: Chromatographic separation has been accomplished using an XTerra C18 Column (150×4.6mm, 5μm particle size) as the stationary phase, with an isocratic system of mobile phase Ammonium dihydrogen phosphate buffer (pH 6.5) and methanol (25:75 proportions) at a flow rate of 1.0 mL/min, detection was executed at 246 nm using an UV detector. The optimized method was validated in accordance to International Conference on Harmonization guidelines. Results: The method developed was found to be linear as regression analysis showed good correlation (R2 = 0.999) with a linear curve at concentration range of 25-150 μg/mL for ALF and 1.25-7.5 μg/mL for DUT. The approach was unique as it was free of degradants in spite of subjecting the drugs to forced degradation. The percentage recovery was in the range of 99.55 and 99.23 % for ALF and DUT respectively from the pharmaceutical dosage form. The developed method showed accurate, precise, robust results with an LOD and LOQ of 0.41 and 0.71 μg/mL and 4.27 and 2.14 μg/mL respectively. Conclusion: Due to the flexibility, accuracy and high precision, the developed method can be employed in routine analysis of bulk and dosage forms. Key words: Alfuzosin Hydrochloride, Dutasteride, RP-HPLC, Method development, Validation, Degradation.

Separation and identification of polyphenol compounds in propolis using offline 2D-RPLC/RPLC coupled with Q-TOF-mass spectrometry

Propolis is a resinous product collected by honey bees from plant-derived materials, with high content of polyphenols associated to various bioactivities. In this study, a powerful separation and identification method of polyphenol compounds from Chinese propolis samples were investigated using off-line two-dimensional reversed-phase liquid chromatography (2D-RPLC/RPLC) in combination with Q-TOF-MS. A Click OEG and an XTerra C18 column were employed for the first and second dimensional separation, respectively. The identification efficacy of 2D-RPLC/RPLC and unidimensional LC (1D-LC) were compared, and 81 components involving 60 polyphenol compounds were characterized by the former method. In contrast, only 36 peaks were detected by the 1D-LC method. The proposed method was demonstrated to be more efficient for the existence or absence of target compounds identification, and that would provide a promising means for accurate determination and quality evaluation of Chinese propolis.

Stability indicating RP-HPLC method for the estimation of flucloxacillin sodium in a tablet dosage form

A Simple, accurate and precise method was developed and validated for the determination of flucloxacillin sodium in its tablet dosage form. The separation was eluted on xterra c18 column (4.6x150mm, 5micron) using a mixture of octane buffer and methanol as mobile phase in a ratio of (30:70) which was pumped through column at a flow rate of 1ml/min. Optimised wavelength for flucloxacillin was 237nm, the retention time was 2.305minutes and the percentage purity was found to be 98.14%. System suitability parameters such as theoretical plate and tailing factor for flucloxacillin sodium was found to be 2991.64 and 1.90 respectively, the proposed method was validated as per ICH guidelines (ICH, Q2 AND (R1)) the method was found to be linear at the concentration range of 20-100µg/ml and the correlation coefficient (r2) value was found to be 0.9994 percentage RSD for precision was 0.9% and percentage RSD for ruggedness was 0.5%. The precision study was precise, robust and repeatable. The LOD and LOQ values are 2.98 and 9.98 respectively. Hence the suggested RP-HPLC method can be used for routine analysis for flucloxacillin sodium in tablet dosage form.

Preparative isolation of arylbutanoid-type phenol [(-)-rhododendrin] with peak tailing on conventional C18 column using middle chromatogram isolated gel column coupled with reversed-phase liquid chromatography.

Reversed-phase liquid chromatography coupled with middle chromatogram isolated gel column was employed for the efficient preparative separation of the arylbutanoid-type phenol [(-)-rhododendrin] from Saxifraga tangutica. Universal C18 (XTerra C18) and XCharge C18 columns were compared for (-)-rhododendrin fraction analysis and preparation. Although tailing and overloading occurred on the XTerra C18 column, the positively charged reversed-phase C18 column (XCharge C18) overcame these drawbacks, allowing for favorable separation resolution, even when loading at a on a preparative scale (3.69mg per injection). The general separation process was as follows. First, 365.0mg of crude (-)-rhododendrin was enriched from 165g Saxifraga tangutica extract via a middle chromatogram isolated gel column. Second, separation was performed on an XTerra C18 preparative column, from which 73.8mg of the target fraction was easily obtained. Finally, the 24.0mg tailing peak of (-)-rhododendrin on XTerra C18 column was selectively purified on the XCharge C18 analytical column. These results demonstrate that the tailing nonalkaloid peaks can be effectively used for preparative isolation on XCharge C18 columns.

Development and validation of an LC-MS/MS method for monitoring larotrectinib, a tropomyosin-related kinase inhibitor, in mouse and human plasma and application to pharmacokinetic studies

Larotrectinib is an orally administered drug and the first and only selective pan-tropomyosin receptor kinase (TRK) inhibitor in clinical development to treat cancer patients harboring a neurotrophic receptor tyrosine kinase gene fusion. In this study, an analytical method to quantify the TRK inhibitor in mouse and human plasma was developed and validated using LC-MS/MS following protein precipitation with acetonitrile. Larotrectinib and an internal standard (carbamazepine) were separated from endogenous substances using an Xterra C18 column with acetonitrile containing 0.1% formic acid as the mobile phase. The ions m/z 429.8 → 342.8 for larotrectinib and m/z 237.0 → 194.0 for carbamazepine detected in multiple reaction monitoring mode were used for the quantitation. The detector response of larotrectinib was linear within the concentration range 5–10,000 ng/mL with a correlation coefficient (r2) of not less than 0.999. The intra- and inter-day precision and accuracy were less than 10.48% and within − 8.99%, respectively, in mouse and human plasma. Larotrectinib was stable under various storage and handling conditions, and no significant matrix effect was observed in both mouse and human plasma. Finally, the assay was successfully applied to the pharmacokinetic study of larotrectinib after its intravenous and oral administration to mice.

LC–MS/MS Determination of Antihypertension Drugs in Rat Plasma and Urine: Applications to Pharmacokinetics

A sensitive, rapid and reproducible LC–MS/MS method for the determination of olmesartan (OLM), amlodipine (ALM) and hydrochlorothiazide (HCZ) in rat plasma and urine has been developed and validated. Irbesartan (IRB) was used as an internal standard. The analytes were separated on a Waters XTerra-C18 column using gradient elution with acetonitrile and 10 mM ammonium acetate buffer (pH 3.5, adjusted with acetic acid) at a flow rate of 1.0 mL min−1. The three analytes were ionized by positive ion electrospray using multiple-reaction monitoring (MRM) mode to monitor precursor → product ion transitions m/z 447.31 → 234.97 for OLM, 408.87 → 238.18 for AML and 290.1 → 204.85 for HCZ. The specificity, matrix effect, recovery, sensitivity, linearity, accuracy, precision, and stabilities were all validated over the concentration range 0.4–100 ng mL−1 for AML, 0.2–100 ng mL−1 for OLM, 0.1–100 ng mL−1 for HCZ. The mean concentrations (Cmax) are 10.32, 587, and 3.4 for OLM, ALM, and HCZ, respectively, by the oral administration of 15 mg kg−1 of each analyte.

Validation of developed method by RP-HPLC for estimation of Prasugrelin human plasma and studying the stability of the drugs in plasma

This paper is concern with a reverse phase high performance liquid chromatography (RP-HPLC) bio-analytical method development and validation for Prasugrel in human plasma using photo diode array detector (PDA detector). The HPLC separation was carried out in an isocratic mode on an X-Terra C18 column (4.6 x 150 mm; 5 μm) with a mobile phase consisting of potassium dihydrogen phosphate [pH 3.0] and acetonitrile in the ratio of 30:70 v/v at a flow rate of 1.0 mL/min. The run time was maintained for 5 mins and the detection was monitored at 210 nm. The percentage recovery was found 99.61-100.06 in human plasma. This reveals that the method is quite accurate. The linearity was found 15-40 μg/mL in human plasma. The inter-day and intra-day precision in plasma was found within the limits. The lower limit of quantification (LLOQ) obtained by the proposed method was 0.05 μg/mL. The percentage relative standard deviation (%RSD) obtained for the drug spiked in plasma for stability studies were less than 2 %.Kathmandu University Journal of Science, Engineering and TechnologyVol. 13, No. 1, 2017, Page: 65-75

Simultaneous determination of dihydrotestosterone and its metabolites in mouse sera by LC-MS/MS with chemical derivatization

Androgens play a vital role in prostate cancer development, and their elimination and blockade are essential in the disease management. DHT is the key ligand for androgen receptor (AR) in the prostate. It is locally synthesized from testosterone. In the prostate, DHT is predominantly metabolized to α-diol and β-diol. Recent studies indicate that impaired DHT catabolism is associated with prostate cancer, signifying the necessity of a sensitive quantitative method for the determination of DHT and its metabolites.In this work, an LC-MS/MS method for the simultaneous quantification of DHT and its metabolites was developed and validated. Steroid-free sera were prepared and used for the preparation of sera calibrators and quality controls (QCs). DHT and its metabolites along with their respective stable heavy isotope labeled analytes representing internal standards were first extracted with methyl tertiary-butyl ether (MTBE) and derivatized with picolinic acid (PA). The derivatized analytes were then extracted again with MTBE, dried under nitrogen and reconstituted in the mobile phase (80% methanol and 0.2% formic acid in water). Baseline chromatographic separation of the derivatized analytes was achieved isocratically on XTerra C18 column (2.1 × 100 mm) using the mobile phase at a flow rate of 0.25 mL/min. Quantitation was performed using multiple-reaction-monitoring mode with positive electrospray ionization. The method has calibration ranges from 0.0500 ng/mL to 50.0 ng/mL for DHT and its two metabolites with acceptable assay precision, accuracy, recovery, and matrix factor. It was applied to the determination of DHT and its metabolites in an animal study.

Development and validation of LC-MS/MS assay for the determination of Butoconazole in human plasma: Evaluation of systemic absorption following topical application in healthy volunteers

Butoconazole is an imidazole antifungal that is more effective than miconazole and clotrimazole for treatment of vaginal candidiasis. A highly sensitive tandem mass spectrometric assay was developed and validated to evaluate systemic absorption of Butoconazole following intravaginal administration. Chromatographic separation was achieved using Waters Xterra C18 column (3µm, 3.0×50.0mm). Liquid-liquid extraction using tert-butyl methyl ether was used for preparation of plasma samples. The mobile phase was solvent A: 0.1% formic acid in water and solvent B: acetonitrile: methanol (30:70, v/v), using gradient elution mode at 0.5mL/min. Detection at positive electrospray ionization in the MRM mode was then employed. Analysis was carried out within 5.5min over a linear concentration range of 0.10–30.00ng/mL. Validation was carried out according to US FDA guidelines for bioanalytical method validation. Matrix effect, recovery efficiency and process efficiency have been investigated for the analyte and internal standard in neat solvent, post-extraction matrix and plasma. The mean percentage recoveries were higher than 80%, the accuracy was 93.51–106.85% and the RSD was below 10% throughout the studied concentration range. Results indicated sufficient stability of the target analyte in plasma at the employed experimental conditions. Results of incurred sample re-analysis and incurred sample stability revealed less than 5% variability. The applicability of the assay for monitoring of the systemic absorption of Butoconazole following intra vaginal application to healthy volunteers was demonstrated. Results confirmed that Butoconazole was detected shortly after intra vaginal administration with Cmax and tmax of 30ng/mL and 6h, respectively.