Sensitivity enhancement and matrix effect evaluation during summation of multiple transition pairs-case studies of clopidogrel and ramiprilat.

Sensitivity enhancement via summation of multiple MRM transition pairs is gaining popularity in tandem mass spectrometric assays. Numerous validation experiments describing the assays for two model substrates, clopidogrel and ramiprilat, were performed. The quantitation of clopidogrel was achieved by the summation of transition pairs m/z 322.2 to m/z 212.0 and m/z 322.2 to m/z 184.0, while that of ramiprilat was achieved by the summation of transition pairs m/z 389.2 to m/z 206.1 and m/z 389.2 to m/z156.1. The use of summation approach achieved sensitivities of >2 fold for both compounds as compared with the reported single MRM transition pair assays. The validation experiments addressed some important assay development issues, such as: (a) lack of impact of matrix effect; (b) unequivocal verification of the percentage contribution of each MRM transition pair towards sensitivity; (c) sensitivity enhancement factor achieved by summation approach of MRM transition pairs; and (d) accurate prediction of quality control samples using summation approach vs a single MRM transition pair. In summary, the appropriateness of using two MRM transition pairs for quantitation was demonstrated for both clopidogrel and ramiprilat. Additionally, pharmacokinetic application of the MRM transition pair assays using a summation approach was established for the two compounds.

A highly sensitive spectrophotometric method for the determination of some phenothiazine antipsychotics using chloramine-T and indigocarmine.

A new simple, accurate and precise spectrophotometric method for the determination of six phenothiazine drugs in pure form and in dosage forms is described. The method is based on the oxidation of the studied drugs by a known excess of Chloramine-T in hydrochloric acid medium and subsequent determination of the unreacted oxidant by reacting it with indigocarmine in the same acid medium. The reacted oxidant corresponds to the drug content. The colored species exhibits maximum absorption at 610 nm. The apparent molar absorptivity values and Sandell sensitivity values are in the range 1.53 x 10(4)-2.96 x 10(4) l mol-1 cm-1 and 13.75-37.15 ng cm-2, respectively. The method is highly sensitive and suitable for 1-15 micrograms ml-1 concentrations with the detection limits being in the range, 0.0651-0.1724 microgram ml-1. The method was successfully applied to the studied drugs in their dosage forms. The results are reproducible within +/- 1% and compare favorably with those obtained by the procedures of the British Pharmacopeia.

Application of potassium dichromate and iron-thiocyanate in the spectrophotometric investigations of phenothiazines.

A spectrophotometric procedure for the determination of phenothiazines in pure form and in a number of their pharmaceutical preparations has been developed that offers the advantages of simplicity, accuracy, precision and sensitivity over many other methods. The method is based on the oxidation of phenothiazines by a known excess amount of potassium dichromate followed by the estimation of unreacted amount of dichromate by reacting with excess of iron(II) and measuring the iron(III) formed by complexing with thiocyanate. The reacted oxidant corresponds to the drug content. Different variables affecting the reaction between drugs and dichromate were studied and optimized. At the maximum absorption of 480 nm, Beer's law is obeyed in the range 2.5-29.75 microg/ml. The molar absorptivity and Sandell sensitivity of the procedure were calculated in addition to detection limit. Statistical treatment of the experimental results indicates that the procedure is precise and accurate. Excipients used as additives in pharmaceutical formulations did not interfere in the proposed procedure. The reliability of the method was established by parallel determination against the official BP methods. The procedure described was successfully applied to the determination of the bulk drugs and in pharmaceutical formulations.

Titrimetric micro-determination of therapeutically active phenothiazines using periodate.

Two new simple, accurate and precise titrimetric micro-procedures are described for the analysis of phenothiazines in pure sample, tablets, injections and syrup using periodate as the oxidant. The first method is based on the oxidation of phenothiazines with periodate in acid medium and the iodate formed in the reaction is determined by reacting it with iodide and titrating the liberated iodine with thiosulfate after masking the excess of periodate with molybdate. In the second procedure, the unreacted (excess) periodate is determined iodometrically under basic conditions. The reaction conditions have been optimised and the stoichiometry of the reaction has been evaluated. A linear relationship exists between the amount of the drug and the titration end-point as shown by the values of correlation coefficient, r (0.9991-0.9999). The slope of the linear relationship has been calculated and found to be in the range, 0.1457-0.3120. The methods were applied to the analysis of dosage forms with results comparable to those given by the official methods. Both the methods are indirect visual titration methods, and are simpler than, and superior to, many existing methods for the assay of phenothiazines.

Measurement and control of dissolved carbon dioxide in mammalian cell culture processes using an in situ fiber optic chemical sensor.

At high viable cell concentrations in large-scale mammalian cell culture processes, the accumulation of dissolved carbon dioxide (dCO(2), typically quantified as an equilibrium gas-phase concentration) becomes problematic as a result of low CO(2) removal rates at reduced surface-to-volume ratios. High dCO(2) concentrations have previously been shown to inhibit cell growth and product formation in mammalian cells and to alter the glycosylation pattern of recombinant proteins. Therefore, reliable monitoring and control of dCO(2) are important for successful large-scale operation. Off-line measurements by instruments such as blood gas analyzers (BGA) are constrained by the low frequency of data collection and cannot be used for on-line control. In a preliminary evaluation of the YSI 8500 in situ sensor, a response time (t(90%)) of 6 min, sensitivity of 0.5% CO(2) (3.6 mmHg), and linearity of measurement (R(2) = 0.9997) between the equivalent gas-phase partial pressure of 0-180 mmHg (0% and 25% CO(2)) were established. Measurements were found to be unaffected by culture pH and typical mammalian cell culture concentrations of glucose, glutamine, glutamate, lactate, and ammonium. The sensor withstood repeated sterilization and cleaning cycles. The reliability of this sensor was demonstrated in microcarrier-based Chinese hamster ovary (CHO) cell perfusion cultures at reactor scales of 30, 40, 340, and 2000 L and was successfully implemented in a dCO(2) control strategy using N(2) sparging.

Quantitation of pharmaceutically important phenothiazines by oxidimetry.

A new spectrophotometric method for the assay of phenothiazines in pure form as well as in pharmaceutical formulations with the chromium(VI)-metol-sulfanilic acid system has been developed. The method is based on the oxidation of the drugs by a known excess of chromium(VI) and subsequent determination of the unreacted oxidant by interacting with metol and sulfanilic acid. The reacted oxidant corresponds to the drug content. The coloured species exhibits maximum absorbance at 530 nm. Beer's law is obeyed over the concentration range 5-60 micrograms ml-1 and the relative standard deviation is found to be less than 2%. The apparent molar absorptivities are in the range 3.77 x 10(3)-3.98 x 10(3) l mol-1 cm-1, the detection limits being in the range 0.6133-1.1349 micrograms ml-1. The method was successfully applied to the determination of the studied drugs in their formulations and the mean percentage recoveries were found to be 97.32-102.80%.

Spectrophotometric determination of ceterizine hydrochloride with Alizarin Red S.

A simple, rapid and sensitive spectrophotometric method has been developed for the assay of ceterizine hydrochloride (CTZH) in bulk drug and its pharmaceutical preparations. This method is based on the ion-pair complex reaction between CTZH and Alizarin Red S in Clarks-Lubs buffer. The chromogen being extractable with chloroform, could be measured quantitatively at 440 nm. All variables were studied to optimise the reaction conditions. Regression analysis of Beer's Law plot showed good correlation in the concentration range 2.5-22 microg ml(-1). The method has a detection limit of 0.1328 microg ml(-1). The proposed method has been successfully applied for the analysis of the bulk drug and its dosage forms such as tablets and syrups. No interference was observed from common pharmaceutical adjuvants.