Human plasma

A linear, precise, accurate and simple reversed-phase high performance liquid chromatography (RP-HPLC) bioanalytical method was developed and validated for determination of sofosbuvir in human plasma (K2EDTA) using ledipasvir as an internal standard. Sofosbuvir and ledipasvir (ISTD) were separated from plasma using solid phase extraction technique and The chromatographic separation was accomplished by using a Zorbax eclipse XDB C18 Column (250×4.6 mm i.d, 5 μm particle size) with 1% orthophosphoric acid (pH6.4) and Acetonitrile in the ratio of 30:70% v/v, as mobile phase in an isocratic elution mode pumped at a flow rate 0.7 mL/min and the column oven temperature was maintained at 25?C. The wavelength selected for quantitation was 254 nm. Retention times of sofosbuvir and internal standard were found to be 3.8min and 7.4min, respectively. The standard curves were found to be linear in the range of 30.566-2000.381 ng/ml for sofosbuvir in human plasma. This method performed an intra-day and inter-day precision within the range of 2.44–11.14 and 5.01–8.70%, respectively. Additional intra-day and inter-day accuracy was within the range of 87.99–105.93% and 94.97–99.18% respectively. Total percentage mean recovery of sofosbuvir from spiked plasma was found to be 86.54%. All the validated parameters were found to be within the Acceptance limit.

A rapid liquid chromatographic tandem mass spectrometry (LC-MS/MS) assay for the measurement of metoclopramide level in human plasma was developed and validated. One ml plasma samples containing metoclopramide and 0.25 µg of loratadine as internal standard (IS) were extracted with 5 ml tert-butyl methyl ether and reconstituted in 80 µl of acetonitrile. Analysis was performed on reversed phase Atlantis dC18 column using a mobile phase of 0.4% formic acid (pH=3.0 ± 0.05) and acetonitrile (20:80, v:v) delivered at a flow rate of 0.25 ml/minute. Analytes were quantified multiple reaction monitoring in positive ion mode with transition mass to charge ratio (m/z) of 299.8→226.9 and 383.4→337.2 for metoclopramide and IS, respectively. Retention times of metoclopramide and IS were around 1.4 and 2.1 minutes, respectively. No significant matrix effect was observed on metoclopramide and IS peaks. Detection limit of metoclopramide in plasma was 0.3 ng/ml. Relationship between metoclopramide level and peak area ratio of metoclopramide / IS was linear (R2 ³ 0.9964) in the range of 0.5–100 ng/ml and inter-day coefficient of variations (CV) and absolute bias were ≤ 12.0% and ≤ 6.0%, respectively. Mean extraction recoveries for metoclopramide and the IS were 91% and 88%, respectively. The method was applied to assess stability of metoclopramide under various conditions generally encountered in the clinical laboratory. Stability of metoclopramide was ≥ 94% and ≥ 95% after 24 hours at room temperature or 48 hours at -20 ºC, respectively, in processed samples and 100% and ≥ 99% after 24 hours at room temperature or 12 weeks at -20 ºC, respectively, in unprocessed samples.

A rapid liquid chromatographic tandem mass spectrometry (LC-MS/MS) assay for the measurement of loratadine level in human plasma was developed and validated. One ml plasma samples containing loratadine and 0.18 µg of metoclopramide as (internal standard, IS) were extracted with 5 ml tert-butyl methyl ether and reconstituted with 80 µl of acetonitrile. Analysis was performed using a reversed phase Atlantis dC18 column and a mobile phase consisting of 0.4% formic acid and acetonitrile (20:80, v:v) and delivered at a flow rate of 0.25 ml/min. The eluents were monitored using electrospray ionization in the positive ion mode with transition mass to charge ratio (m/z) at 383.4→337.2 and 299.8→226.9 for loratadine and IS, respectively. The retention times of the IS and loratadine were around 1.53 and 2.33 min, respectively. Mean matrix effect was measured as -11.4% for loratadine and -14.4% for the IS. Detection limit of loratadine in plasma was 0.3 ng/ml. The relationship between loratadine concentration in plasma and the peak area ratio of loratadine / IS was linear (R2 ³ 0.9945) in the range of 0.5–100 ng/ml, and the intra- and inter-day coefficient of variations (CV) were ≤ 11.3%. Mean extraction recoveries for loratadine and the IS were 87% and 91% respectively, whereas accuracy (relative recovery) ranged from 99% to 111% quality control samples and from 93% to 105%  using back- calculated concentrations. The method was applied to assess the stability of loratadine under various conditions generally encountered in the clinical laboratory. Stability for processed samples (24 hours at room temperature, 48 hours -20 ºC) and unprocessed samples (24 hours at room temperature, 12 weeks -20 ºC) was ≥ 94%. Key words: Loratadine, Metoclopramide, Human plasma, HPLC  

A simple, precise, and rapid high performance liquid chromatography (HPLC) method for the determination of ketoconazole level in human plasma using itraconazole as an internal standard (IS) was developed and validated. 0.25 ml plasma samples containing ketoconazole were mixed with 15 µg of the IS. After adding 0.25 ml acetonitrile, the mixture was vortexed for two minutes and then centrifuged for 10 minutes at 16000 rpm at room temperature. The clear supernatant was transferred into an auto-sampler vial, and 100 µl was injected into the HPLC system with a run time of 10 min. The compounds of interest were efficiently separated on 4.6 x 150 mm, Symmetry Shield TMRP18 5-µm steel column, using a Guard Pak pre-column module with    Radial-Pak C18 5-µm insert, and detected using Waters 2475 multi λ fluorescence detector with an the excitation and emission wavelengths set at 260 and 375 nm, respectively. The mobile phase consisted of 0.02 M potassium dihydrogen phosphate (pH = 6.0, adjusted with 0.1 M sodium hydroxide) and acetonitrile (40:60, v:v), and was delivered at a flow rate of 1.0 ml/min. No interference from blank plasma or commonly used drugs was observed; and the detection limit of ketoconazole was 0.1 µg/ml. The relationship between ketoconazole concentration in plasma and peak area ratio of ketoconazole /IS was linear (r2 ≥ 0.9979) in the range of 0.1– 20 µg/ml. Intra- and inter-day coefficients of variation (CV) were ≤ 8.1%  and  ≤ 9.7%, respectively, with corresponding biases of ≤ -13% and ≤ 0.9%, respectively. Mean extraction recovery of ketoconazole and IS were ≥ 85% and 92%, respectively. Using the method, ketoconazole was found to be stable for 48 hrs at -20°C (≥ 95%) in processed samples and for 8 weeks at -20°C (100%) in unprocessed samples. Key words: Ketoconazole, Itraconazole, Human plasma, HPL

A simple, precise, and rapid high performance liquid chromatography (HPLC) method for the determination of acetaminophen level in human plasma using caffeine as an internal standard (IS) was developed and validated. 0.5 ml plasma samples containing acetaminophen were mixed with 50 µg of the IS. After adding 30 µl of 50% perchloric acid, the mixture was vortexed for one minute and then centrifuged for 5 minutes at 13200 rpm. The clear supernatant was transferred into an auto-sampler vial and 100 µl was injected into the HPLC system with a run time of 7.0 min. The compounds of interest were efficiently separated on Symmetry C18 (4.6 x 150 mm, 5-µm) column, and were detected with a photodiode array detector set at 245 nm. The mobile phase consisted of water, methanol, and acetonitrile (80:10:10, v:v:v) and was delivered at a flow rate of 0.9 ml/min. No interference in blank plasma or by commonly used drugs was observed; and the detection limit of acetaminophen was 0.05 µg/ml. The relationship between acetaminophen concentration in plasma and peak area ratio of acetaminophen /IS was linear (r2 ≥ 0.9991) in the range of 0.1– 40 µg/ml. Intra- and inter-day coefficient of variations (CV) and biases were ≤11.6%  and  ≤10.8%, and ≤≤14.0 and ≤12.8, respectively. Extraction recovery of acetaminophen and the IS from the plasma samples was ≥99% and 86%, respectively. Using the method, acetaminophen was found to be stable under conditions generally encountered in the clinical laboratory (≥99% and 91% in processed and unprocessed samples, respectively). Further, the method was successfully used to measure acetaminophen level in plasma samples from a healthy volunteer.

A simple and precise reversed-phase high performance liquid chromatography (HPLC) method for the determination of meloxicam in human plasma was developed and validated. Using piroxicam as an internal standard (IS), separation was achieved on Symmetry shield RP-18 column. 0.5 ml plasma samples were prepared by protein precipitation using trifluoroacetic acid and acetonitrile. The mobile phase consisted of 0.025 M dibasic potassium phosphate (pH=6.0, adjusted with phosphoric acid), methanol, and acetonitrile (73:5:22, v:v:v) and was delivered at a flow rate of 1.5 ml/min. Eluents were measured using photodiode array detector set at 355 nm. Under these conditions, no interference in blank plasma or of commonly used drugs was observed. The relationship between the concentration of meloxicam in plasma and peak height ratio of meloxicam to the IS was linear over the range of 0.05-2.0 μg/ml. Intra-day and inter-day coefficient of variations (CV) and biases were ≤ 6.0% and ≤ 8.6%, and ±5.9% and ±5.3%, respectively. Extraction recovery of meloxicam and the IS from plasma was ≥80% and 98%, respectively. The method was applied to assess the stability of meloxicam under various clinical laboratory conditions. In processed samples, meloxicam was stable for at least 24 hours at room temperature (≥ 82%) and 48 hours at -20C (≥ 95%). In unprocessed sample it was stable for at least 24 hours at RT (≥ 82%), 16 weeks at -20ºC (≥ 87%), and after three freeze-thaw cycles (≥ 90%). The method is suitable for clinical and bioavailability investigation involving meloxicam concentration in the therapeutic range.

A simple and precise reversed-phase high performance liquid chromatography (HPLC) method for the determination of hydrochlorothiazide (HCT) in human plasma was developed and validated. Using hydroflumethiazide as an internal standard (IS), separation was achieved on Atlantis dC 18 column. The mobile phase, 10 mM monobasic potassium phosphate and acetonitrile (80:20, v: v), was delivered at a flow rate of 1.2 ml/min. The eluent was monitored by photodiode array detector, with the wavelength set at 272 nm. Plasma samples containing HCT and IS were extracted with methyl tert butyl ether and reconstituted in mobile phase. No interference in blank plasma or of commonly used drugs was observed. The relationship between the concentration of HCT in plasma and peak area ratio of HCT to the IS was linear over the range of 5-300 ng/ml. The intra-day and inter inter-day coefficient of variation and bias were

A rapid and sensitive LC-MS/MS method for the quantification of nebivolol using d4- nebivolol as internal standard has been developed and validated. The nebivolol and d4- nebivolol were extracted by liquid- liquid extraction using tert-butyl ethyl ether and separated on Kromasil 100-5C 4.6x100mm column using a mixture of 0.1% formic acid in 5 mM ammonium acetate, methanol and acetonitrile at composition of (20:20:60 v/v) at a flow rate of 0.5 mL/min. Detection involved an API-4000 LC-MS/MS with electrospray ionization in the positive mode.  The method was validated as per the FDA guidelines and shown to provide an intra and inter day precision and accuracy within the acceptable limit with in a run time of 3.0 min. The proposed method can adopt for the regular bioequivalence study analysis and also can easily adoptable for clinical drug monitoring due to its simplicity and ruggedness.

In this paper the authors proposed a simple, sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay method for the determination of mosapride in human plasma. This method employed mosapride–d5 as an internal standard (IS). Analyte and the IS were extracted form 100 µL of human plasma using solid–phase extraction with no drying, evaporation and reconstitution steps. The chromatographic separation was achieved on a C18 column by using a mixture of methanol and 5mM ammonium acetate (80:20, v/v) as the mobile phase at a flow rate of 1.0 mL/min. The linearity of the method was established in the concentration range 0.18–60.0 ng/mL with r2 ³ 0.99. The intra–day and inter–day precision (%CV) and accuracy results in five validation batches across five concentration levels were well within the acceptance limits. The validated method was found to be applicable to clinical studies.

A well developed and validated RP – HPLC method by UV- detection was used for the determination of Gatifloxacin in human plasma with metronidazole as internal standard. Solid phase extraction was involved in the process. The drug and the internal standard were eluted under isocratic mode using a 150 X 4.6 mm i.d, 5 µm Phenomenex ODS 2 C18 column. The mobile phase composed of a mixture of 5:95 % v/v methanol and 20mM mixed phosphate buffer (pH 3.5± 0.05) at a flow rate of 1.4 mL/Minute. The detection wave length of the detector was 268 nm. A volume of  50 µL was injected and the runtime of the method was 9 minutes. The method showed good linearity in the range of  50.1 to 7000.9 ng/mL. The recovery of gatifloxacin was 92.42 % with a standard deviation of 1.533 and recovery of internal standard was 87.6 %. The LOD of Gatifloxacin was 50.1 ng/mL. Matrix effects were not observed.