A guide for using experimental design in chromatographic method development: applied to the analysis of selected anti-diabetic pharmaceutical combinations.

A guide experimental design in chromatographic method development was described and applied successfully to the analysis of different recently approved anti-diabetic pharmaceutical combinations. Enhancement of UHPLC analysis of alogliptin benzoate either with pioglitazone hydrochloride or with metformin hydrochloride was achieved. The optimal chromatographic conditions were not attained by trial and error that requires a large number of experiments. Alternatively, a computer program was used as a systematic optimization strategy for the design of the experiment which accurately predicts the combined effect of different factors simultaneously. Resolution between peaks was studied by the proposed fractional factorial design approach performed by the Minitab® Program using screening and optimization steps. Application of the central composite design was implemented. A Pareto chart was used to exclude the insignificant variables. Linearity ranges were found to be 0.5-40 µg ml-1, 1-20 µg ml-1 and 1-32 µg ml-1 for alogliptin benzoate, pioglitazone hydrochloride and metformin hydrochloride, respectively. The proposed method is applicable for the analysis of six pharmaceutical dosage forms namely, Nesina®, Actos®, Glucophage®, Oseni®, Kazano® and Actoplus MET® tablets.

Validated spectrophotometric methods for determination of some oral hypoglycemic drugs.

Four accurate, precise, rapid, reproducible, and simple spectrophotometric methods were validated for determination of repaglinide (RPG), pioglitazone hydrochloride (PGL) and rosiglitazone maleate (RGL). The first two methods were based on the formation of a charge-transfer purple-colored complex of chloranilic acid with RPG and RGL with a molar absorptivity 1.23 × 103 and 8.67 × 102 l•mol-1•cm-1 and a Sandell's sensitivity of 0.367 and 0.412 µg•cm-2, respectively, and an ion-pair yellow-colored complex of bromophenol blue with RPG, PGL and RGL with molar absorptivity 8.86 × 103, 6.95 × 103, and 7.06 × 103 l•mol-1•cm-1, respectively, and a Sandell's sensitivity of 0.051 µg•cm-2 for all ion-pair complexes. The influence of different parameters on color formation was studied to determine optimum conditions for the visible spectrophotometric methods. The other spectrophotometric methods were adopted for demtermination of the studied drugs in the presence of their acid-, alkaline- and oxidative-degradates by computing derivative and pH-induced difference spectrophotometry, as stability-indicating techniques. All the proposed methods were validated according to the International Conference on Harmonization guidelines and successfully applied for determination of the studied drugs in pure form and in pharmaceutical preparations with good extraction recovery ranges between 98.7-101.4%, 98.2-101.3%, and 99.9-101.4% for RPG, PGL, and RGL, respectively. Results of relative standard deviations did not exceed 1.6%, indicating that the proposed methods having good repeatability and reproducibility. All the obtained results were statistically compared to the official method used for RPG analysis and the manufacturers methods used for PGL and RGL analysis, respectively, where no significant differences were found.

The cardiovascular safety of triphasic contraceptive steroids.

It was hypothesized that estrogen-induced cardioprotection is mediated by up-regulation and down-regulation of expression of nitric oxide (NO) and P-selectin, respectively. Published data on circulating levels of the vasodilator NO, atherogenic glycoprotein P-selectin, and lipoprotein-a [Lp(a)] in users of triphasic contraceptive steroids are lacking. A total of 30 healthy women (nonusers, controls) and 82 women using oral triphasic contraceptive steroids (ethinyl estradiol and levonorgestrel: Triovlar, Schering AG) for 18 to 24 cycles participated in this study. Fasting blood samples were obtained from users and nonusers for the determination of P-selectin and Lp(a) by enzyme immunoassay and NO by a colorimetric method. The serum Lp(a) levels in OC users were significantly higher than those of nonusers. On the other hand, the serum NO levels in OC users were significantly elevated when compared to nonusers. Plasma P-selectin was significantly lowered in OC users p < 0.005. These results demonstrate the beneficial effects of ethinyl estradiol in the triphasic contraceptive regimen. Ethinyl estradiol may afford a degree of anti-atherogenic-cardioprotective effect by up-regulation of the expression of the vasodilator NO and down-regulation of the expression of the atherogenic P-selectin. This may outweigh the cardiovascular risk of the increased atherogenic Lp(a). This study may explain the very low rate of mortality from venous thromboembolism in OC users, which compares favorably with the risks that many people accept in daily life.

Serum interleukin-2 and soluble interleukin-2 receptor in gestational trophoblastic diseases.

OBJECTIVE: To investigate serum interleukin-2 (IL-2) and soluble interleukin-2 receptor (SIL-2) levels in gestational trophoblastic diseases (GTD). METHODS: Sixty-six patients with GTDs and 23 first-trimester healthy pregnant women (controls) participated in this study. According to the World Health Organization scoring system, GTDs were subgrouped into the following groups: 30 hydatidiform mole spontaneous regression (HMSR), 12 postmolar gestational trophoblastic tumors of high risk (PMHR), 14 low-risk choriocarcinomas, and ten high-risk choriocarcinomas. Before treatment, a blood sample from each case was assayed for beta-hCG by radioimmunoassay, IL-2 by IRMA, and SIL-2R by enzyme-linked immunosorbent assay. Follow-up beta-hCG assays were carried out at weekly intervals after treatment for 3 months, then monthly for 1 year. RESULTS: Serum IL-2 levels in all subgroups of GTD were significantly lower than that of controls. Meanwhile, there were concomitant significant elevations of serum SIL-2R, showing mean rises of 3.86-fold, 3.9-fold, twofold, and 6.1-fold for cases of HMSR, PMHR, low-risk choriocarcinoma, and high-risk carcinoma, respectively. All cases of high-risk choriocarcinoma revealed abnormally high SIL-2R values. There was a significant positive correlation between serum beta-hCG and SIL-2R concentrations. CONCLUSION: The possible causes of IL-2 decreases and SIL-2R increases may indicate a defective immune response in GTDs. The high correlation between SIL-2R level and tumor burden suggests the use of serum SIL-2R assay for disease monitoring: SIL-2R is indirect marker of tumor activity, and it is useful in the differential diagnosis of GTD because a normal value of serum SIL-2R excludes high-risk cases of choriocarcinoma.

Serum tumour necrosis factor alpha levels in benign and malignant lesions of the endometrium in postmenopausal women. A preliminary study.

Tumour necrosis factor alpha (TNF alpha) concentration was determined by a solid phase immunoradiometric assay in sera of 20 healthy postmenopausal women (reference group), 10 women suffering from postmenopausal bleeding with histologically confirmed cystic glandular hyperplasia, and 10 postmenopausal bleeding patients with histologically confirmed endometrial adenocarcinoma. In cases of endometrial adenocarcinoma serum TNF alpha (157.6 +/- 28 pg/ml) was found to be significantly higher than in controls (80.6 +/- 4.1 pg/ml). The rate of abnormally high values of serum TNF alpha increased with advancing stage of the disease. On the other hand, serum TNF alpha level in cases of endometrial hyperplasia (38 +/- 6 pg/ml) was significantly lower than in healthy individuals. It seems that the rise of serum TNF alpha in cases of endometrial carcinoma represents a possible mechanism of immune surveillance. The results suggest clinical usefulness of serum TNF alpha estimations for the differential diagnosis of benign and malignant lesions of the endometrium in women with postmenopausal bleeding.

Plasma beta-endorphin level in cases of luteal phase defect.

OBJECTIVE: To evaluate beta-endorphin secretion in euprolactinemic cases of luteal phase defect (LPD). DESIGN: Serial blood samples from the 18th to the 26th day of the menstrual cycle were assayed for beta-endorphin, progesterone (P), estradiol (E2), and prolactin (PRL) in cases of LPD and controls. Diagnosis of LPD was based on determinations of serum P and premenstrual endometrial biopsy. SETTING: From Cairo University Hospitals. PATIENT, PARTICIPANTS: Twenty-six women with LPD and 8 normal fertile women (controls) were chosen. INTERVENTIONS: None. MAIN OUTCOME MEASURES: beta-Endorphin, P, E2, and PRL concentrations were determined by the corresponding 125I radioimmunoassay. RESULTS: Plasma beta-endorphin level in cases of LPD varied from 2.58 to 9.14 pmol/L, whereas the level of controls varied from 2.41 to 5.57 pmol/L. The mean value of plasma beta-endorphin in cases of LPD was significantly higher than that of controls by 146% (P less than 0.0005). In spite of the significant decrease of serum P in cases of LPD, serum E2 level did not differ significantly from that of controls. CONCLUSION: The possible sources of beta-endorphin rise and its implication in the etiology of LPD are explained.

Assessment of left ventricular function in schistosomiasis.

Inspite of the fact that the haemodynamic changes in the right ventricle in schistosomal disease received proper attention, left ventricular changes were not equally assessed. That the left ventricle could suffer in bilharzial disease is a possibility, probably due to the development of shunts and toxic myocarditis. In this wor5, left ventricular functions were studied using the rate of maximum rise of intraventricular pressure (kp/dt) as an index of contractility. The study included 10 normal controls, 5 cases with bilharzial liver fibrosis and no ascites or cor pulmonale, 10 cases with BLF, and ascites but no cor pulmonale, and 10 cases with bilharzial cor pulmonale. Diminished dp/dt values were obtained in 8 out of the 10 cases of bilharzial cor pulmonale, in whom the left ventricular pressure curve also showed distortion and delay in the descending limb of the isometric relaxation phase, denoting defective contractility of the left ventricle, and probably a diastolic volume overload in these cases. In BLF with or without ascites, the dp/dt values were surprisingly increased possibly due to increased catecholamines in these cases secondary to ineffective destruction in the liver suffering from parenchymatous disease. The left ventricle is thus shown to suffer in bilharzial disease particularly in cases with bilharzial cor pulmonale.