Rajaa F Hussein

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 cefazolin level in human plasma using ceftriaxone as an internal standard (IS) was developed and validated. 0.25ml plasma samples containing cefazolin were mixed with 17.5 µg of the IS. After adding 0.3 ml of cold methanol kept in fridge at 4 °C, the mixture was vortexed for two min and then centrifuged for 15 min 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 18.0 min. The compounds of interest were efficiently separated on 4.6 x 150 mm Atlantis dC18-5µm steel column, using a Guard Pak pre-column module with Nova-Pak C185-µm insert, and detected using Waters 2998 photodiode array detector set at 270 nm. The mobile phase consisted of a mixture of 0.02 M of cetyltriethyl ammonium bromide and 0.01 M dipotassium hydrogen phosphate (pH = 6.5, adjusted with phosphoric acid) and acetonitrile, 60:40 (v:v) for 6 min, 50:50 for 6 min, and 60:40 for 6 min. It was delivered at a flow rate of 1.5 ml/min. No interference in blank plasma or by commonly used drugs was observed; and the detection limit of cefazolin was 0.1 µg/ml. The relationship between cefazolin concentration in plasma and peak area ratio of cefazolin/IS was linear (r2 ≥ 0.9979) in the range of 0.2–200 µg/ml. Intra- and inter-day coefficient of variation (CV) was ≤ 7.4%  and  ≤ 5.7%, with the corresponding bias of ≤ 4.9% and ≤ 5.1%. Mean extraction recovery of cefazolin and IS were 97%, respectively. Using the method, cefazolin was found to be stable in processed samples for 48 hrs at -20°C ( ≥ 96%), and in unprocessed samples for 8 weeks at -20°C (≥ 98%), Further, the method was successfully used to measure cefazolin level in plasma samples.