Ahmed S. Fayed

Separation and determination of clopidogrel and its impurities by capillary electrophoresis.

Clopidogrel bisulphate, an anti-platelet drug, has been separated from its impurities, namely impurity A, B and C by capillary zone electrophoresis (CZE) using uncoated fused-silica capillary (50.0 microm internal diameter, 31.2cm total length). Four factors affected the separation: buffer concentration, pH of the buffer, concentration of the chiral selector and the applied voltage. Optimization and robustness studies were performed with the aid of reduced central composite experimental design. The buffer used was triethylamine-phosphoric acid and the chosen chiral selector was sulphated beta-cyclodextrin (SCD). The best separation was achieved by using 10mM buffer, pH 2.3, containing 5% (mass/volume (m/v)) SCD. Reversed polarity mode was used with an applied voltage of -12kV and the capillary temperature was maintained at 20 degrees C. The method was validated for quantitative determination of the drug. It offered a limit of detection (LOD) of 0.13 microg/ml, a limit of quantitation (LOQ) of 0.4 microg/ml, and a linearity range of 0.4-300 microg/ml. Commercial bulk samples were analyzed using the developed method.

A five-year analysis of the incidence of glomerulonephritis at Cairo University Hospital-Egypt

Our study aimed to obtain a comprehensive review of the incidence of biopsy-proven glomerulonephritis (GN) at the Cairo University Hospitals, Egypt, over the last five years. We analyzed the clinical and pathological data of all renal biopsy samples that were performed during the period from July 2003 to July 2008. Renal biopsy samples of 924 patients were referred for pathological assessment during the period of the study [437 male and 487 female patients; their mean age was 26.5 ± 14.6 years (range: 2.5-71 years)]. Focal segmental glomerulo-nephritis was the most frequent cause of primary GN (21.21%), followed by mesangial proliferative GN (18.93%), diffuse proliferative GN (13.96%), focal proliferative GN (12.77%) and membranous GN (10.93%). The results could be explained by the high incidence of lupus nephritis among the study subjects as well as the relatively young age of the study group.

A novel spectral resolution and simultaneous determination of multicomponent mixture of Vitamins B1, B6, B12, Benfotiamine and Diclofenac in tablets and capsules by derivative and MCR–ALS

A novel method was developed for spectral resolution and further determination of five-component mixture including Vitamin B complex (B1, B6, B12 and Benfotiamine) along with the commonly co-formulated Diclofenac. The method is simple, sensitive, precise and could efficiently determine the five components by a complementary application of two different techniques. The first is univariate second derivative method that was successfully applied for determination of Vitamin B12. The second is Multivariate Curve Resolution using the Alternating Least Squares method (MCR-ALS) by which an efficient resolution and quantitation of the quaternary spectrally overlapped Vitamin B1, Vitamin B6, Benfotiamine and Diclofenac sodium were achieved. The effect of different constraints was studied and the correlation between the true spectra and the estimated spectral profiles were found to be 0.9998, 0.9983, 0.9993 and 0.9933 for B1, B6, Benfotiamine and Diclofenac, respectively. All components were successfully determined in tablets and capsules and the results were compared to HPLC methods and they were found to be statistically non-significant.

Chromatographic analysis of multicomponent mixture of vitamins B1, B6, B12, benfotiamine and diclofenac; part II: LC-tandem MS/MS method for simultaneous quantification of five components mixture in pharmaceutical formulations and human plasma

A novel high performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was established for the simultaneous determination of multivitamins, namely thiamine hydrochloride (B1), pyridoxine (B6), cyanocobalamin (B12) and benfotiamine (BEN) and their co-formulated drug, diclofenac sodium (DIC) using torsemide as internal standard (IS). Chromatographic separation was accomplished on Shimadzu LC-20 AT Series HPLC, equipped with Sunfire C18 column (Waters) (50 × 4.6 mm, 5 μm) using methanol : 0.02 M ammonium acetate (80 : 20, v/v) pH 6.0 for B1, B12, BEN and DIC, while using methanol : acetonitrile : 0.01 M ammonium formate (80 : 10 : 10, v/v/v) pH 6.25 for B6 on separate runs at a flow rate of 1.0 mL min−1. Sample preparation involves extraction with methanol using 30 ng mL−1 of IS. Electrospray ionization (ESI) source was operated in the positive ion mode. The multiple reaction monitoring (MRM) mode on a triple quadrupole mass spectrometer was used to quantify the drugs utilizing the transitions of 265.07/122.10, 170.01/152.20, 678.64/147.01, 467.18/122.10, 296.02/214.20, 348.99/263.90 (m/z) for B1, B6, B12, BEN, DIC and IS, respectively. The proposed method was effectively applied for the analysis of laboratory prepared mixtures, in spiked human plasma as well as in their combined pharmaceutical formulations. A detailed analytical and bioanalytical validation was conducted in compliance with the FDA and ICH guidelines proving the method to be selective, linear, precise and accurate over the concentration ranges of 0.01–20.00, 0.02–50.00, 15.00–500.00, 5.00–500.00 and 10.00–500.00 ng mL−1 for the five compounds, in order. The simplicity and sensitivity of this method allows its use in the quality control of the cited drugs.

Simultaneous Determination of Cinchocaine Hydrochloride and Betamethasone Valerate in Presence of Their Degradation Products

Cinchocaine hydrochloride (CIN) and betamethasone valerate (BMV) are co-formulated in pharmaceutical formulations that could be used for local treatment of hemorrhoids. Both drugs are susceptible to hydrolytic degradation. Two sensitive and precise stability-indicating chromatographic methods were developed for the simultaneous determination of both active pharmaceutical ingredients. The developed methods were applied for quantitation of CIN and BMV in their pure forms, in presence of their corresponding degradation products and in their pharmaceutical formulation. The first method was a high performance liquid chromatographic (HPLC) one, separation and quantitation was achieved using a Waters Spheriosorb® 5 μm ODS2 C18 analytical column and an isocratic mobile phase formed of acetonitrile-acetate buffer (pH 6.5 ± 0.1) in a ratio of (55:45, v/v). The mobile phase was pumped at a flow rate of 1.2 mL/min. UV-detection was done at 240 nm using photodiode array detector. The second method was based on thin layer chromatography (TLC) fractionation coupled with densitometric determination. Separation was done on high performance thin layer chromatography (HPTLC) silica gel 60F254 plates using a developing system formed of chloroform-toluene-ethanol-acetic acid in a ratio of (4.5:4.5:1:1, by volume). The separated bands were scanned densitometrically at 240 nm. For the HPLC method, linearity was confirmed over concentration ranges of 4-300 and 4-350 μg/mL for CIN and BMV, respectively. For the HPTLC-densitometric method, the obtained ranges were 0.5-12 and 0.5-10 μg/band for CIN and BMV, respectively. The developed methods were optimized and validated according to the ICH guidelines. CIN acid degradation products were separated and identified by mass spectroscopy. The developed HPLC method was used to study the kinetics of acid and alkali degradation of the both drugs. The results obtained were statistically analyzed and compared with those obtained by applying the official methods for both drugs.

Chromatographic Determination of Cyclopentolate Hydrochloride and Phenylephrine Hydrochloride in the Presence of Their Potential Degradation Products.

Two sensitive, selective, and precise stability-indicating methods have been developed for the simultaneous determination of the active pharmaceutical ingredients cyclopentolate hydrochloride (CLO) and phenylephrine hydrochloride (PHE) in their pure forms and in the presence of their degradation products. The methods were applied for the determination of CLO and PHE in a pharmaceutical formulation. Method A was based on isocratic elution HPLC determination. Separation was achieved using a Waters Spherisorb ODS2 C18 analytical column (5 μm particle size) and a mobile phase of 0.1% heptane-1-sulphonic acid sodium salt in methanol-water (80 + 20, v/v). The flow rate was 1.0 mL/min and detection was performed at 210 nm. Method B was an HPTLC- densitometric method using HPTLC silica gel 60 F254 plates and an optimized mobile phase of ethyl acetate-methanol-ammonia (8 + 2 + 0.1, v/v/v). The separated spots were densitometrically scanned at 210 nm. Polynomial equations were used for regression. The developed methods are suitable for the determination of CLO and PHE in their binary mixture and in the presence of their corresponding degradation products. The two methods were validated in compliance with International Conference on Harmonization guidelines and successfully applied for the determination of CLO and PHE as synthetically prepared in laboratory mixtures and in the presence of their possible degradation products. CLO alkaline degradation products were stated as potential impurities in British Pharmacopoeia. The degradation products were separated and identified by mass spectra. Postulation of a PHE oxidative degradation pathway was suggested. The obtained results were statistically analyzed and compared with those obtained by applying the official methods for both drugs.

The incidence of biopsy-proven glomerulonephritis in Cairo University, Egypt: a 5-year study

Sir, The incidence of biopsy-proven glomerulonephritis (GN) varies in different geographical areas and is affected by socio-economic conditions, race, differences in genetic susceptibility and environmental exposure. Recent studies suggested a changing pattern of incidence of GN in different parts of the world [1,2]. For instance, the incidence of end-stage renal disease (ESRD) as a result of focal segmental glomerulosclerosis (FSGS) has increased 11-fold in the past two decades in a recent study [2]. Our study aimed to obtain a comprehensive review of the incidence of biopsy-proven glomerulonephritis in Cairo University, Egypt, over the last 5 years. We analysed the clinical and pathological data of all renal biopsy samples that were obtained during the period from July 2003 to 2008. Age, gender, indication of renal biopsy and the pathological findings were recorded for analysis. A total of 924 renal biopsy samples were referred for pathological assessment during the period of the study. The monthly incidence of biopsy-proven GN was 15.4 (range 13–19). Proliferative GN was reported in 497 cases (53.78%) and non-proliferative GN was reported in 427 cases (46.22%). Lupus nephritis was reported in 264 cases (28.57%). The female/male ratio was 221/41, 70% of lupus patients aged 18–45 years and 85% had renal impairment (mean serum creatinine was 3.21 ± 4.09 mg/dl). The common glomerular pathologies in patients with lupus nephritis were the proliferative classes II–IV (28.78, 30.30, 27.65%, respectively). Morphologically, FSGS was the most frequent cause of GN (21.21%) followed by mesangial proliferative GN (18.93%), diffuse proliferative GN (13.96%), focal proliferative GN (12.77%) and membranous GN (10.93%) (Table ​(Table1).1). In females, mesangial proliferative, focal proliferative GN and diffuse proliferative GN were the predominant pathological findings, and in males FSGS, diffuse proliferative GN and mesangial proliferative GN were predominant. In those aged <18, mesangial proliferative GN, minimal change disease and FSGS were more prevalent compared to adults. Figure ​Figure11 shows the frequency of GN in patients with renal insufficiency. Fig. 1 Incidence of GNs in patients with renal insufficiency [352 cases (38.09%)]. We conclude that FSGS and proliferative GN (SLE and others) were the predominant forms of GN in the population of the study. Compared to other Arab countries, FSGS was the predominant ... Table 1 Incidence of biopsy proven GNs in the study group Conflict of interest statement. None declared.

Therapeutic Plasma Exchange Outcomes in Cairo University Hospitals: 6 Years Experience: Cairo University Plasma Exchange Outcome

Therapeutic plasma exchange is used in treating different immunological and non‐immunological diseases. We analyzed the outcome of 308 patients treated by 1783 membrane plasma exchange sessions from January 2011 until January 2017 at Cairo University Hospital. Thrombotic microangiopathies were the commonest indication [73 (23.7%) patients] with response in 63/73 patients (86.3%), followed by systemic vasculitis with pulmonary‐renal involvement [40(13%) patients] with recovery in 32/40 patients (80.0%), Guillain‐Barré syndrome [39(12.7%) patients] with recovery in 30/39 patients (76.9%), myasthenia gravis [31(10.1%) patients] with response in 26/31 patients (83.9%), and catastrophic antiphospholipid syndrome [28(9.1%) patients] with recovery in only 6/28 patients (21.4%). Complications included hypotension [276/1783 (15.5%) sessions], hypocalcemia [26/308 (8.5%) patients], and 37/308 (12%) patients died. Sepsis caused mortality in 29/37 (78.4%) of patients. In conclusion, our therapeutic plasma exchange experience shows a favorable outcome for thrombotic microangiopathies, systemic vasculitis, myasthenia gravis, and Guillain‐Barré syndrome. Sepsis was the leading mortality cause.

Fibroblast growth factor-23 is a strong predictor of insulin resistance among chronic kidney disease patients

Abstract Insulin resistance (IR) is very common among chronic kidney disease (CKD) patients. Disturbance in mineral and bone metabolism (MBD) seems to play a role in the pathogenesis of insulin resistance. Fibroblast growth factor-23 (FGF23) is evolving as the most important link between MBD and many pathologic sequences of CKD. The aim was to evaluate IR in pre-dialysis CKD patients looking for a possible association to mineral metabolism among CKD patients. A total of 100 stage 3–5 CKD patients were selected beside 20 normal control subjects. Homeostatic model assessment of insulin resistance (HOMA-IR) was used to assess IR in selected cases. Both groups were compared for fasting blood glucose (FBG), fasting blood insulin (FBI), HOMA-IR, estimated glomerular filtration rate (eGFR), serum calcium (Ca), phosphorus (P), 25 hydroxy vitamin D (25 OH vit D), parathormone (PTH), and uric acid (UA). Correlation study between HOMA_IR and different studied parameters was performed. HOMA-IR is significantly higher in CKD (8.87 ± 3.48 vs. 3.97 ± 0.34 in CKD vs. control, respectively, p < .001). In addition CKD patients have significantly higher FGF23 (235 ± 22.96 vs. 139 ± 12.3 pg/mL, p < .001), PTH (76.9 ± 15.27 vs. 47.9 ± 2.52 pg/mL, p < .001), P (4.3 ± 0.67 vs. 3.6 ± 0.23 mg/dL, p < .001), and UA (5 ± 1.22 vs. 4.85 ± 0.48 mg/dL, p < .001) and significantly lower Ca (8.2 ± 0.3 vs. 8.9 ± 0.33 mg/dL, p < .001), and 25 (OH) vit D (17 ± 5.63 vs. 37 ± 3.43 ng/mL, p < .001). Stepwise linear regression analysis revealed that BMI, GFR, Ca, P, and FGF23 were the only significant predictors of HOMA IR. Increased IR in CKD is a consequence of the uremic status and is intimately associated with disturbed phosphate metabolism and FGF23. Further studies are needed to look for an underlying mechanism.

Incidence and Characteristics of de novo Renal Cryoglobulinemia After Direct-Acting Antivirals Treatment in an Egyptian Hepatitis C Cohort

Introduction: The side effects profile of the new direct-acting antivirals for the treatment of hepatitis C virus (HCV) is not fully elucidated. Objective: In this cross-sectional study, we aim to describe the incidence and characteristics of a novel observation of de novo renal cryoglobulinemic glomerulonephritis after successful treatment with DAA. Methodology: A total of 12,985 Hepatitis C Patients (genotype IV) received the new DAA. After successful treatment, patients with deranged renal functions or proteinuria were referred to the nephrology department for assessment. The clinical manifestations ranged from lower limb edema to the development of purpura skin lesions. Cryoglobulins were tested in the serum using the PCR detection. Results: Fifty patients had detectable de novo cryoglobulins in the serum. The most common type in renal biopsies was membranoproliferative glomerulonephritis (52%) and chronic kidney disease (CKD) developed in 46% of cases. Conclusion: De novo cryoglobulinemic glomerulonephritis and progression to CKD may rarely complicate successful treatment of HCV using direct-acting antivirals.