Reproducible Reversed-phase High-performance Liquid Chromatography Research Articles

VALIDATION OF STABILITY INDICATING RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF GLECAPREVIR AND PIBRENTASVIR BY USING ANALYTICAL QUALITY BY DESIGN (AQBD) METHOD

The quantitative measurement of Glecaprevir and Pibrentasvir has been created using a simple, quick, precise, sensitive, and reproducible reverse-phase high-performance liquid chromatography (RP-HPLC) method. It is more difficult to analyse varying amounts of pharmaceutical active medicinal ingredients in dosage forms without interferences. Therefore, the objective of the current work is to estimate Glecaprevir and Pibrentasvir simultaneously by adopting an Analytical Quality by Design (AQbD), a rotatable central composite-based technique using RP-HPLC-based method development and validation. Glecaprevir and Pibrentasvir were separated by chromatography using a Kinetex RP-18 Column (100x4.6mm, 2.6µ) column and a mobile phase made up of Acetonitrile: 0.1% tri fluoro acetic acid in a ratio of 26.464:73.536 v/v. The flow rate was 1.0 ml/min, and a photodiode array detector operating at room temperature was used to detect absorption at 236 nm. ICH criteria have been used to validate the offered techniques' linearity, accuracy, precision, and other attributes. The degradation study's findings showed that the medications deteriorated in high-stress situations. The chemical and pharmaceutical sectors might easily implement this unique AQbD-based analytical technique for routine analysis without any regulatory constraints.

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF RP-HPLC FOR AMLODIPINE BESYLATE AND LISINOPRIL IN COMBINED TABLET DOSAGE FORM BY USING SIMULTANEOUS ESTIMATION METHOD


 
 
 The purpose of this study was to develop and validate a simple, sensitive, accurate, and reproducible reverse phase high performance liquid chromatography (RP-HPLC) method for simultaneous estimation of amlodipine besylate and lisinopril in a Tablet dosage form. The chromatographic measurement was performed on a Phenomenex C18 (250 × 4.6 mm, 5um) column with an optimised Acetate buffer mobile phase: Methanol (65:35). The flow rate was one ml/min, and the detecting wavelength was 221 nm. Triethanolamine was used to adjust the pH to 5. In the concentration range of 4-20 ug/ml, amlodipine besylate and lisinopril showed a linear response of the suggested approach. The correlation coefficients ('r' values) for amlodipine besylate and lisinopril were 0.9998 and 0.9995, respectively, and the retention times were 3.279 for amlodipine besylate and 6.124 for lisinopril, respectively. The developed chromatographic technique was validated for specificity, linearity, precision, accuracy, LOD, and LOQ using ICH Q2(R1) criteria. The analysis results have been validated in accordance to ICH guidelines
 
 

Development and validation of a high-performance liquid chromatography method for the estimation of esomeprazole in bulk and tablet dosage form

Introduction: An accurate, simple, precise, rapid, economic and reproducible reverse-phase high-performance liquid chromatography method was developed and validated for the estimation of Esomeprazole (ESO) in bulk and tablet dosage form. Method: The separation was carried out on Finepak SIL C18T-5 column (250 × 4.6 mm, 5.0 µm i. d.) using potassium dihydrogen phosphate buffer (0.025M): ACN (20:80 v/v) and at a flow rate of 1.0 mL/min. using UV detector at 302 nm with a run time of 10 min. The method was validated for accuracy for linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ) and robustness. Results: The standard calibration curve was linear with R2 = 0.995. LOD and LOQ obtained for esomeprazole were 0.0001 and 0.0004 µg/mL respectively. The method was found robust for possible changes. Results of analysis of other parameters were also tested and validated as per ICH guidelines and recovery studies confirmed the accuracy of the proposed method. validation studies showed that the developed HPLC method is simple, reproducible, rapid, precise and reliable. The high recovery and low relative standard deviation confirm the suitability of the developed method for the determination of esomeprazole in the tablet dosage form. Conclusion: This method may be used as a more convenient and efficient option for the analysis of esomeprazole to establish the quality of the substance during routine analysis with consistent and reproducible results.

RP-HPLC Method Validation for Estimation of Tenofovir Disoproxil Fumarate in Pharmaceutical Oral Dosage Form

A simple, efficient and reproducible Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) method for the estimation of Tenofovir Disoproxil Fumarate in Pharmaceutical formulation has been validated. The Separation was carried out on C-18 column 4.6 mm x 250 cm x 5 µm column at ambient temperature using Buffer: Acetonitrile in the ratio of 60: 40 as a mobile phase. The flow rate was 1 ml/min and effluent was detected at 260nm. The retention times was found to be 3 mts. The percentage recovery was within the range between 99.23% to 101.44 %. The method was linear in the range of 50%-150% for Tenofovir Disoproxil Fumarate where r2 = 0.999. The percentage relative standard deviation for accuracy and precision was found to be less than 2%. The method was validated according to ICH guidelines and the acceptance criteria for specificity, linearity, accuracy, precision, ruggedness and robustness were met in all cases in detecting Tenofovir Disoproxil Fumarate. Hence the method could be successfully applied for routine analysis of Tenofovir Disoproxil Fumarate in the solid dosage form.

Quantification and Standardization of Andrographolide in Andrographis Paniculata Samples by Validated RP-HPLC and HPTLC Methods.

Andrographis paniculata (family Acanthaceae) is known as Kalmegh, one of the traditionally used important medicinal plant contains several biologically active phytochemical including andrographolide. A. paniculata is broadly used by healthcare practitioners in India and also used in different traditional medicinal system. In this study, the leaves of A. paniculata were collected from West Medinipur, East Medinipur, South 24 Parganas, Purulia and Hooghly district of West Bengal, India. This study aiming towards validation and development of a simple, precise and reproducible reverse-phase high-performance liquid chromatography (RP-HPLC) and high-performance thin layer chromatography (HPTLC) methods for quantification of andrographolide in A. paniculata extracts. The validated RP-HPLC and HPTLC study confirmed that different concentrations of andrographolide content present in the plant samples, which are collected from above different districts of West Bengal, India. The amounts of andrographolide were found to be 2.71% (w/w), 3.19% (w/w), 1.83% (w/w), 1.73% (w/w) and 2.94% (w/w) in RP-HPLC study and 2.13% (w/w), 2.51% (w/w), 1.01% (w/w), 1.25% (w/w) and 2.15% (w/w) in HPTLC study. This precise, reproducible, accurate and specific method can be used for the quantification of andrographolide in kalmegh, as per the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines recommendations.

Method Development and Validation of Gallic Acid in Liquid Dosage Form by Using RP-HPLC Method.

A simple, rapid, precise, sensitive, and reproducible reverse-phase high-performance liquid chromatography (RP-HPLC) method has been developed for the quantitative analysis of gallic acid in the pharmaceutical dosage form. Chromatographic separation of gallic acid was achieved on Waters Alliance-e 2695, by using Waters X-Terra RP-18 (150×4.6mm, 3.5μ) column and the mobile phase containing 0.1% formic acid and ACN in the ratio of 70:30% v/v. The flow rate was 1.0mL/min; detection was carried out by absorption at 275 nm using a photodiode array detector at ambient temperature. The number of theoretical plates and tailing factor for gallic acid was NLT 2000 and should not be more than 2 respectively. Percentage relative standard deviation of peak areas of all measurements is always less than 2.0. The proposed method was validated according to ICH guidelines. The method was found to be simple, economical, suitable, precise, accurate, and robust method for quantitative analysis of gallic acid.

Development and Validation of Stability Indicating Assay for Simultaneous Determination of Bupivacaine and Meloxicam in Bulk and Pharmaceutical Formulations by Using Rp-Hplc Method

A simple, rapid, precise, sensitive, and reproducible reverse phase high-performance liquid chromatography (RPHPLC) method has been developed for the quantitative estimation of Bupivacaine and Meloxicam in pharmaceutical dosage form. Aim and objective of our study is to determine the amount of bupivacaine and meloxicam by using RP-HPLC using Waters alliance HPLC system, Quaternary gradient pump of e2695 series equipped with an auto sampler injector with 10μl is injected, and eluted with the mobile phase containing Acetonitrile and Water in the ratio of 60:40 v/v which is pumped at a flow rate of 1 ml/min and detected by UV detector at 225 nm. The peak of Bupivacaine and Meloxicam was eluted at retention times of 2.646 min and 3.136 min, respectively. Chromatographic separation of Bupivacaine and Meloxicam was achieved on Waters Alliancee2695by using Chiralcel ODH 150x4.6mm, 5μcolumn and the mobile phase containing Acetonitrile and water in the ratio of 60:40% v/v. The flow rate was 1.0 ml/min; detection was carried out by absorption at 225 nm using a photodiode array detector at ambient temperature. The number of theoretical plates and tailing factor for Bupivacaine and Meloxicamwere NLT 2000 and kept not more than two respectively. Peak areas, Percentage relative standard deviation of all measurements always less than 2.0. This method was validated according to ICH guidelines. The method was a simple, economical, suitable, precise, accurate & robust method for quantitative analysis of Bupivacaine and Meloxicam study of its stability.

Bioanalytical Method Development and Validation of Selinexor in Rat Plasma by Liquid Chromatography-Tandem Mass Spectrometry

A simple, rapid, precise, sensitive, and reproducible reverse-phase high-performance liquid chromatography (RPHPLC) LCMS/MS method has been developed for the bioanalytical method for Selinexor with D6-Selinexor as Internal Standard in the pharmaceutical dosage form. Chromatographic separation of Selinexor was achieved on Waters Alliance-e2695, by using X-Bridge phenyl, 150x4.6mm, 3.5μm column, and the mobile phase containing 0.1% Formic acid & Acetonitrile in the ratio of 80:20% v/v. The flow rate was 1.0 ml/min; detection was carried out by absorption at 225nm using a photodiode array detector at ambient temperature. The method was validated to fulfill International Conference on Harmonization (ICH) requirements and this validation included specificity, selectivity, matrix effect, linearity, the limit of detection (LOD), limit of quantification (LOQ), precision, and accuracy. The proposed method was Bio-analytical validated according to USFDA guidelines. This method was found to be a very simple, economical, suitable, precise, accurate, and stable method for pharmacokinetic analysis of Selinexor and study of its stability. The calibration curve was linear over the concentration range from 0 to 40 ng/ml, and the lower limit of detection of 12.5 ng/ml. The accuracy and precision of the method were within the acceptable limit of ±20% at the lower limit of quantitation and ±15% at other concentrations. Selinexor was unstable at room temperature it showed more than 25% loss after 24 h. While, Selinexor is very stable at refrigerator 40C auto-sampler, freeze/thaw cycles, and 30 days storage in a freezer at 35 ± 20C. All results were acceptable and this confirmed that the method is suitable for its intended use in routine quality control and an assay of drugs.

RP-HPLC method development and validation for simultaneous estimation of telmisartan, rosuvastatin calcium, and amlodipine besylate in combination.

Dyslipidemia-hypertension proves to be a major risk factor for cardiovascular diseases. In order to achieve better adherence and cost-effectiveness than free equivalent combination therapies, a fixed-dose combination therapy with telmisartan (TEL), rosuvastatin calcium (ROS) and amlodipine besylate (AML) is required in this type of patients.

Selective SGLT2 Inhibitor’s Estimation and Validation from Galenical Form by RP-HPLC Method

Aim: The recent analysis is required to do novel and simple, sensitive, precise, efficient, instant and reproducible reverse phase high performance liquid chromatography (RP-HPLC) method for estimation of antidiabetic drug in the unit dosage form. Validation of this method is also planned to make it suitable for the actual use.
 Study Design: Department of Pharmaceutical Chemistry, Datta Meghe Institute of Medical Sciences deemed to be university, Wardha in collaboration with Balkh university, Mazar-e-sharif, Afghanistan between August 2021 and December 2021.
 Methodology: In that article develop the method and validate it by estimation of antidiabetic drugs in solid dosage form by RP-HPLC, by using System suitability test, Repeatability, Precision studies (Intra-day and Interday/Intermediate), Linearity/Calibration studies, Robustness, Force degradation, Specificity, Drug recovery/accuracy studies.
 Results: as per ICH guidelines, the performance if system suitability in remogliflozin were achieved all guidelines; in that, tailing factor (T),separation factors(α),theoretical plates(N),capacity factor (k’), resolution (R) and RSD (%). The validated stress degradation studies under thermal, oxidative, alkali and acid, in few degradation products for remogliflozin (REM).
 Conclusion: From the results we conclude that, this novel technique which validated for exploration by reverse phase high performance liquid chromatography (RP-HPLC) should be used for routine quality control of remogliflozin (REM) prediction from developed formulation.

Analytical Method Development and Validation for the Estimation of Silodosin and Dutasteride in Capsule Dosage Form by RP-HPLC Method

A simple, rapid, accurate, efficient and reproducible reverse phase high performance liquid chromatography (RPHPLC) method was developed and subsequently validated for simultaneous estimation of silodosin and dutasteride capsules. The analysis was carried out using Unison C8 column (250 mm x 4.6, 5 μm particle size) at 40°C temperature using mobile phase buffer:Methanol:Acetonitrile (20%:40%:40% v/v/v) at flow rate 1.0 ml/min. Quantification was achieved with PDA detector at a wavelength of 275nm. The silodosin peak retention time was 2.4 mins and the dutasteride retention was 10.9 mins, so the total run time was set to 14mins. The linearity range of Silodosin and Dutasteride is 20-120 μg/ml and 1.25-7.5 μg/ml respectively. Linear regression found to be 0.999. The proposed method was validated according to International Conference on Harmonisation (ICH) guidelines. The results shown in the method was accurate, precise, sensitive and economical. Thus, the current study showed that the developed reverse-phase liquid chromatography method is sensitive and selective for the estimation of Silodosin and Dutasteride in Capsule Dosage Form

RP-HPLC Method Development and Validation for the Estimation of Mirabegron in Bulk and Dosage Form

A new, simple, precise, accurate and reproducible Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method for Simultaneous estimation of bulk and pharmaceutical formulations. Separation of Mirabegron was successfully achieve , C18, 250X4.6mm, 5µm or equivalent in an isocratic mode utilizing methanol water (70:30) at pH 5.0 Adjusted to OPA at a flow rate of 1.0ml/min and eluate was monitored at 243nm, with a retention time of 2.584 minutes for Mirabegron. The method was validated and the response was found to be linear in the drug concentration range of 50µg/ml to150 µg/ml for Mirabegron. The values of the correlation coefficient were found to 0.999for Mirabegron. The Limit of Detection(LOD) and Limit of Quantification (LOQ) for Mirabegron were found to be 0.149 and 0.498 respectively. This method was found to be good percentage recovery were found to be 99 indicates that the proposed method is highly accurate. The specificity of the method shows good correlation between retention times of standard with the sample so, the method specifically determines the analyte in the sample without interference from excipients of tablet dosage forms. The method was extensively validated according to International Council for Harmonisation(ICH) guidelines for Linearity, Accuracy, Precision, Specificity and

A new analytical method for determination of tenofovir disoproxil fumarate and emtricitabine in pharmaceutical formulations by RP-HPLC method

A simple, rapid, precise, sensitive and reproducible reverse phase high performance liquid chromatography (RP-HPLC) method has been developed for the quantitative analysis of Tenofovir Disoproxil Fumarate and Emtricitabine in pharmaceutical dosage form. Chromatographic separation of Tenofovir Disoproxil Fumarate and Emtricitabine was achieved on Waters Alliance -2695, by using Luna C18 (250mm x 4.6mm, 5µm) column and the mobile phase containing 0.1% TEA adjusted pH-2.5 with OPA & ACN in the ratio of 60:40 v/v. The flow rate was 1.0 ml/min, detection was carried out by absorption at 261 nm using a photodiode array detector at ambient temperature. The number of theoretical plates and tailing factor for Tenofovir Disoproxil Fumarate and Emtricitabine were NLT 2000 and should not more than 2 respectively. The linearity of the method was excellent over the concentration range 30-450 µg/ml and 20-300 µg/ml for Tenofovir Disoproxil Fumarate and Emtricitabine respectively. The correlation coefficient was 0.999. % Relative standard deviation of peak areas of all measurements always less than 2.0. The proposed method was validated according to ICH guidelines. The method was found to be simple, economical, suitable, precise, accurate & robust method for quantitative analysis of Tenofovir Disoproxil Fumarate and Emtricitabine study of its stability.

A new analytical method for determination of dolutegravir and rilpivirine in pharmaceutical formulations by RP-HPLC method

A simple, rapid, precise, sensitive and reproducible reverse phase high performance liquid chromatography (RP-HPLC) method has been developed for the quantitative analysis of Dolutegravir and Rilpivirine in pharmaceutical dosage form. Chromatographic separation of Dolutegravir and Rilpivirine was achieved on Waters Alliance -2695, by using Luna C18 (250mm x 4.6mm, 5µm) column and the mobile phase containing 0.1% OPA & ACN in the ratio of 50:50 v/v. The flow rate was 1.0 ml/min, detection was carried out by absorption at 245 nm using a photodiode array detector at ambient temperature. The number of theoretical plates and tailing factor for Dolutegravir and Rilpivirine were NLT 2000 and should not more than 2 respectively. The linearity of the method was excellent over the concentration range 10-150 µg/ml and 5-75 µg/ml for Dolutegravir and Rilpivirine respectively. The correlation coefficient was 0.999. % Relative standard deviation of peak areas of all measurements always less than 2.0. The proposed method was validated according to ICH guidelines. The method was found to be simple, economical, suitable, precise, accurate & robust method for quantitative analysis of Dolutegravir and Rilpivirine study of its stability.

CLEANING VALIDATION OF A SIMPLE AND RAPID REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR THE SIMULTANEOUS ESTIMATION OF ASPIRIN AND ROSUVASTATIN

Objective: This study describes a new, simple, precise, accurate, and reproducible reversed-phase high-performance liquid chromatography (RP-HPLC) cleaning validation method for simultaneous estimation of rosuvastatin and aspirin.
 Methods: The proposed RP-HPLC method was carried out on AGILENT-ZORBAX RP-Inertsil column (250 mm × 4.6 mm, 5 μm) in an isocratic mode utilizing potassium dihydrogen phosphate buffer (pH 2.5 with OPA):acetonitrile (50:50,v/v) as mobile phase, at a flow rate of 1.5 ml/min. Detection was carried out at 243 nm using UV detector.
 Results: The method was found specific as there was no swab interference. The Beer–Lambert’s law was obeyed in the concentration range of 0.5–20 μg/ml for both rosuvastatin and aspirin. The mean percentage recoveries at 100% level were 89.4% for rosuvastatin and 82.1% for aspirin. The limit of detection and limit of quantification for rosuvastatin and aspirin were 0.03 μg/ml and 0.1 μg/ml, respectively. The method was found to be robust and precise with percentage RSD <2.0%.
 Conclusion: A simple, novel, and economical RP-HPLC method for cleaning validation has been developed for the simultaneous estimation of rosuvastatin and aspirin. The method was validated as per ICH guidelines for specificity, linearity, accuracy, precision, and robustness. The developed method can be used as a sensitive analytical tool for ensuring the effectiveness of the cleaning procedure adopted.

A selective LC-MS/MS method for simultaneous quantification of Artemether, Lumefantrine and their principle metabolites in human plasma

We have developed and validated a sensitive, selective and reproducible reversed-phase high-performance liquid chromatography method coupled with electrospray ionization mass spectrometry (HPLC–ESI-MS/MS) for the simultaneous quantitation of artemether (ART), dihydroartemisinin (DHA), lumefantrine (LUM) and desbutyl-lumefantrine (DBL) in human plasma. Mefloquine was used as an internal standard (IS). The analytes were extracted by protein precipitation procedure and separated on a reversed-phase Zorbax SB-Ciano column with a mobile phase composed of acetonitrile and 20mM aqueous ammonium formate containing 0.5% (v/v) formic acid. Multiple reaction monitoring was performed in the positive ion mode using the transitions m/z 316.3→m/z 163.1 (ART), m/z 302.3→m/z 163.1 (DHA), m/z 530.3→m/z 512.2. (LUM), m/z 472.2→m/z 454.1 (DBL) and m/z 379.1→m/z 361.1(MQ) to quantify the drugs. Calibration curves in spiked plasma were linear (r2 ≥ 0.9992) over the range of 5–1500 ng/mL for ART/ DHA and 5–5,000 ng/mL for LUM/DBL. The lower limit of quantitation (LLOQ) was 10 ng/mL ART/ DHA and 5 ng/mL for LUM/ DBL. The mean R.S.D. values for the intra-run precision were 2.2% , 3.8%, 1.9% and 4.7% and for the inter-run precision were 3.2%, 3.6% , 4.4% and 3.5% for ART, DHA, LUM and DBL, respectively. The mean percentage recovery values were 93.2%, 98.5%, 97.1% and 99.4% for ART, DHA, LUM and DBL, respectively. No matrix effect was detected for all the analytes and the IS. The validated method was successfully applied to determine the plasma concentrations of ART, DHA, LUM and DBL in pregnant and non-pregnant women volunteers in a multiple-dose pharmacokinetics study over the course of 336 hours.

LC–MS/MS Method for Quantitation of Hydrochlorothia-zide and Nifedipine in Human Plasma

We have developed and validated a novel, sensitive, selective and reproducible reversed-phase high-performance liquid chromatography method coupled with electrospray ionization mass spectrometry (HPLC–ESI-MS/MS) for the simultaneous quantitation of hydrochlorothiazide (HCTZ) and nifedipine (NFP) from only 200 µL of human plasma. Diazepam (DZP) was used as an internal standard (IS). The analytes were extracted by a liquid-liquid extraction procedure with ethyl acetate-dichloromethane and separated on a reversed-phase Polaris 5 C18 Aanalytical column using a mobile phase composed of methanol containing 0.1% (v/v) formic acid and 5mM aqueous ammonium formate pH 6.0, delivered at a flow-rate of 300µL/min. Multiple reaction monitoring was performed in the negative ion mode using the transition m/z296.1→m/z205.2 (HCTZ), positive ion mode transitions m/z347.2→m/z 315.1 (NFP) and m/z285.0→m/z 193.2 (DZP) to quantify the drugs. Calibration curves in spiked plasma were linear (r2 ≥ 0.9953) from 5–2000 ng/mL for HCTZ and 5 – 400 ng/mL for NFP with a lower limit of quantification (LLOQ) of 5 ng/mL for both drugs. The intra- and inter- assay precisions (coefficient of variation) were less than 9% and the mean extraction recoveries were 98.1% (HCTZ), 99.5% (NFP) and 93.8% for the IS (DZP). The validated method was successfully applied to a limited population pharmacokinetics study of HCTZ and NFP among patients on hypertension care.

DEVELOPMENT AND VALIDATION OF STABILITY INDICATING RP-HPLC METHOD FOR ESTIMATION OF SATRANIDAZOLE FROM ITS FORMULATION

A sensitive stability-indicating high performance liquid chromatography (HPLC) method was developed and validated for quantitative estimation of satranidazole (SAT), a new nitroimidazole with potent antiamoebic activity and accessible in market as tablet and dry syrup either alone or in combination with ofloxacin. The present study involves the development of simple, accurate, precise, reproducible reversed phase high performance liquid chromatography (RP-HPLC) method for determination of satranidazole from its formulation. Isocratic elution at a flow rate of 1.0 mL/min was employed on Hemochrom Intsil C-18 (250 mm× 4.6 mm, 5 ?m) column at 25°C. The mobile phase consists of acetonitrile: double distilled water (DDW), pH 4 adjusted with acetic acid in the ratio of 90: 10 V/V. The UV detection wavelength was 320 nm, and 20 ?L sample was injected. The retention time for SAT was about 3.9 minutes. Forced degradation studies for stability-indicating method (SIM) were conducted as per the presented conditions of ICH guidelines. The drug showed instability in alkaline and oxidation condition while it remained stable in heat, acid and photolytic conditions. According to ICH guidelines, the developed method was validated for the following parameters: linearity, accuracy, precision, specificity, robustness, limit of detection (LOD) and limit of quantification (LOQ). The method was found to be accurate, linear, precise, robust, economical and stability-indicating for analysis of the drug as well as marketed formulation.

Liquid chromatography-tandem mass spectrometry for the simultaneous quantitation of ceftriaxone, metronidazole and hydroxymetronidazole in plasma from seriously ill, severely malnourished children.

We have developed and validated a novel, sensitive, selective and reproducible reversed-phase high-performance liquid chromatography method coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS/MS) for the simultaneous quantitation of ceftriaxone (CEF), metronidazole (MET) and hydroxymetronidazole (MET-OH) from only 50 µL of human plasma, and unbound CEF from 25 µL plasma ultra-filtrate to evaluate the effect of protein binding. Cefuroxime axetil (CEFU) was used as an internal standard (IS). The analytes were extracted by a protein precipitation procedure with acetonitrile and separated on a reversed-phase Polaris 5 C18-Analytical column using a mobile phase composed of acetonitrile containing 0.1% (v/v) formic acid and 10 mM aqueous ammonium formate pH 2.5, delivered at a flow-rate of 300 µL/min. Multiple reaction monitoring was performed in the positive ion mode using the transitions m/z555.1→ m/z396.0 (CEF), m/z172.2→ m/z 128.2 (MET), m/z188.0→ m/z125.9 (MET-OH) and m/z528.1→ m/z 364.0 (CEFU) to quantify the drugs. Calibration curves in spiked plasma and ultra-filtrate were linear ( r 2 ≥ 0.9948) from 0.4-300 µg/mL for CEF, 0.05-50 µg/mL for MET and 0.02 - 30 µg/mL for MET-OH. The intra- and inter- assay precisions were less than 9% and the mean extraction recoveries were 94.0% (CEF), 98.2% (MET), 99.6% (MET-OH) and 104.6% (CEF in ultra-filtrate); the recoveries for the IS were 93.8% (in plasma) and 97.6% (in ultra-filtrate). The validated method was successfully applied to a pharmacokinetic study of CEF, MET and MET-OH in hospitalized children with complicated severe acute malnutrition following an oral administration of MET and intravenous administration of CEF over the course of 72 hours.

LC–MS/MS Method for Quantitation of Hydrochlorothia-zide and Nifedipine in Human Plasma

We have developed and validated a novel, sensitive, selective and reproducible reversed-phase high-performance liquid chromatography method coupled with electrospray ionization mass spectrometry (HPLC–ESI-MS/MS) for the simultaneous quantitation of hydrochlorothiazide (HCTZ) and nifedipine (NFP) from only 200 µL of human plasma. Diazepam (DZP) was used as an internal standard (IS). The analytes were extracted by a liquid-liquid extraction procedure with ethyl acetate-dichloromethane and separated on a reversed-phase Polaris 5 C18 Aanalytical column using a mobile phase composed of methanol containing 0.1% (v/v) formic acid and 5mM aqueous ammonium formate pH 6.0, delivered at a flow-rate of 300µL/min. Multiple reaction monitoring was performed in the negative ion mode using the transition m/z296.1→m/z205.2 (HCTZ), positive ion mode transitions m/z347.2→m/z 315.1 (NFP) and m/z285.0→m/z 193.2 (DZP) to quantify the drugs. Calibration curves in spiked plasma were linear (r2 ≥ 0.9953) from 5–2000 ng/mL for HCTZ and 5 – 400 ng/mL for NFP with a lower limit of quantification (LLOQ) of 5 ng/mL for both drugs. The intra- and inter- assay precisions (coefficient of variation) were less than 9% and the mean extraction recoveries were 98.1% (HCTZ), 99.5% (NFP) and 93.8% for the IS (DZP). The validated method was successfully applied to a limited population pharmacokinetics study of HCTZ and NFP among patients on hypertension care.