Development and validation of a simple method for the determination of Atorvastatin calcium in pure and pharmaceutical formulations using spectrofluorimetry.

A simple, accurate, precise, sensitive and selective spectrofluorimetric method was developed and validated for the determination of Atorvastatin calcium (ATV), an HMG-CoA reductase inhibitor, in its pure and tablet dosage form. The proposed method was based on direct measurement of the native fluorescence of ATV. Fluorescence analysis was accomplished by using an emission wavelength 385 nm after excitation at the wavelength of 270 nm in acetonitrile, without difficult preparation steps of the sample solution such as separation, extraction, pH adjustment or derivatization. All variables affecting the fluorescence intensity such as measurement time, temperature, and diluting solvent were investigated and optimized. Under the typical conditions, a validation study for linearity, range, accuracy, precision, selectivity and robustness of the proposed method was implemented according to ICH guidelines. The fluorescence intensity was linear over concentration range of (0.4-12) µg/ml (r = 0.9999), and the lower limits of detection and quantification were 0.079 and 0.24 µg/ml, respectively. Good accuracy and precision results were obtained through using the presented method with excellent mean recovery value 100.08 ± 0.32 which was in the acceptable range (98.0-102.0%), and RSD <2%, proving the precision of the developed method. Specificity was proved in the presence of excipients and Amlodipine besylate (AML) which encountered usually as combined drug with ATV. The developed method was successfully applied to the analysis of pharmaceuticals containing the mentioned drug with no interference from other drugs or dosage form additives, and the recoveries were in the range of 99.11 ± 0.75 to 100.89 ± 0.70. Furthermore, the obtained results were compared with reported HPLC method. Then, the t- and F- values were calculated and compared with the theoretical ones, which indicate good precision and high accuracy of the proposed method. Therefore, this method is valuable, reliable, and very suitable to be applied in routine quality control laboratories.

Evaluation of new formulations for neutralizing antimicrobial preservatives in pharmaceutical preparations.

Background: Neutralization of preservative systems is essential to obtain reliable results when testing samples containing preservatives such as nutritional, cosmetic and pharmaceutical products. Therefore, the aim of this study was to prepare and investigate the neutralization ability of in-house neutralizing systems made of available cost-effective materials in the inactivation of preserved pharmaceutical suspensions. Ibuprofen and Cefpodoxime proxetil preserved suspensions were chosen as the quenching model since subsequent microbiological studies will be conducted on their local pharmaceutical formulations available in the Syrian market. Methods: We reported toxicity and efficacy ratios of ten neutralizing systems (No.1 to No.10) containing polysorbate 80, cetomacrogol 1000 and polyoxyl 40 hydrogenated castor oil with various concentrations dedicated to the inactivation of Ibuprofen and Cefpodoxime proxetil preserved suspensions, methyl paraben/propyl paraben mixture and sodium benzoate controls against low inoculum ranging between 1 × 102-1.2 × 103 CFU of five challenged bacteria and fungi; Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans and another environmental isolate of Aspergillus niger. Neutralizing systems validation was conducted according to USP chapter 1227 criteria to assess the acceptance of recovery comparisons for both "Neutralizing systems toxicity test" and "Neutralizing systems efficacy test" which enabled determining the appropriate neutralizing formula for both neutralization of preservative system of a specified product and being non-toxic towards the challenged microorganism additions. Results: Most neutralizing formulas used in the study were non-toxic for all tested microorganisms. According to "Neutralizing systems efficacy test", No. 3 (polysorbate 80 (3%)) and No. 10 (polysorbate 80 (1%), cetomacrogol 1000 (1%) and polyoxyl 40 hydrogenated castor oil (1%)) effectively recovered at least three microorganisms when used in the neutralization of samples. Most limitations were observed when neutralizing ibuprofen suspension. However, we found neutralizing system No. 10 against Pseudomonas aeruginosa, No. 3 and No. 5 against Escherichia coli and No. 8 and No. 10 against Candida albicans were effective in the neutralization of ibuprofen suspension. All neutralizing systems effectively inactivated the preservative system of cefpodoxime proxetil suspension using all microorganisms while several neutralizing systems failed in quenching cefpodoxime proxetil suspension against Staphylococcus aureus. Conclusion: Due to the variation in the neutralization efficacy relative to the product sample and challenged microorganism, neutralization validation procedure must be undertaken before microbiological testing of pharmaceuticals which makes the development and validation of neutralizing systems an essential procedure.

A New method for simultaneous qualitative and quantitative determination of amlodipine besylate and atorvastatin calcium in bulk and pharmaceutical formulations using transmission FT-IR spectroscopy.

A simple, precise, rapid, and eco-friendly FTIR spectrophotometric method was developed and validated for simultaneous analysis of amlodipine (AML) and atorvastatin (ATV) in drug combination preparations. Firstly, synthetic mixtures were made and scanned with FTIR instrument. Then the result spectra were converted automatically to absorbance spectra. The calibration model was made depending on Beer's law which relates concentration to absorbance. Two characteristic bands corresponding to the carbonyl group at 1708-1688 cm-1 and 1660-1632 cm-1 for AML and ATV, respectively, were selected for quantification. The absorbance of a series of standards was measured as the AUC of the chosen bands. Then, the calibration line was obtained by plotting the measured AUCs and the actual concentrations against each other. Validation tests were performed per ICH recommendations. Specificity was evaluated by the separation of APIs and excipients from marketed preparations by methanol. Then, the spectra of extracted excipients, APIs, and pharmaceutical samples were taken and overlapped. The selected peaks were specific and did not interfere with each other or other peaks from the excipients used in the tablet's matrix. Linearity for AML and ATV was in the range of 0.1-1% w/w with excellent coefficients of determination (R2), 0.998 and 0.999 for AML and ATV, respectively. The proposed analytical method was accurate and precious, as the RSD values were less than 2%. The proposed FTIR method was successfully applied to estimate the exact quantity of APIs in pharmaceutical samples. Recoveries were accepted in accordance with USP and were in the range of 94.62-100.6% and 98.175-101.06% for AML and ATV, respectively. Likewise, the acquired results were compared with the HPLC method. And the t- and F- tests were calculated and compared with the theoretical values, which indicate the similarity of results in both developed and reported methods.

Microbial evaluation of some syrups in Syrian pharmacies.

Microbial contamination of syrups can bring clinical hazards to patients as well as physical and chemical changes in the product. Aims: Studying the influence of the war on the Syrian pharmaceutical industry from a microbiological point of view by assessing the microbiological quality of syrup samples taken from Syrian pharmacies. Methodology: Fifty different syrups from 29 different companies having various manufacture dates were collected during validity period between 9-2019 and 6-2021 in Aleppo, Syria. Membrane filtration technique was performed to quantify microbial contamination of the collected syrup samples. This involved passing the samples through filter nitrocellulose membrane disks with a pore size of 0.45 µm then transferring the filter disks alongside any collected microorganisms into Tryptone Soya Agar, Sabouraud Dextrose agar, Xylose lysine Deoxycholate agar and Eosin Methylene Blue agar plates. Colonies observed on these plates were counted and the number of viable microbes in the original sample was expressed as colony forming units per milliliter (CFU/mL). Investigation of Escherichia coli in all syrup samples and Salmonella in herbal syrup samples was also performed. Results: This study revealed that 28 syrups (56%) had no growth of either bacterial or fungal colonies; 33 syrups (66%) had no growth of bacterial colonies; 43 syrups (86%) had no growth of fungal colonies. On the other hand, 7 syrups (14%) exceeded the pharmacopoeial limit for bacterial growth and 6 syrups (12%) exceeded that for fungal growth. Furthermore, 5 syrup samples (10%) were on the high permissible limits for bacterial contamination and none for fungal contamination.All syrups were free from E. coli and all herbal syrups were free from Salmonella. Taken together, out of the fifty syrups examined 13 syrups (26%) exceeded the pharmacopoeial limits and therefore pharmacopoeial accepted syrups constitute a percentage of (74%). Conclusion: Although the majority of samples tested showed compliance with the pharmacopoeial limits of microbiological contamination, the small proportion of syrups in the Syrian market exceeding the pharmacopoeial limit is still concerning and reveals the implications of post-war conditions on the quality of manufacturing in the Syrian pharmaceutical industry. That said, it remained within the proper limits compared to studies conducted in other countries in similar situations.This study, therefore, highlights the need to apply the Good Manufacturing Practice (GMP) rules more strictly in order to limit microbiological contamination in pharmaceutical syrups to ensure the quality of products and safety of users. We suggest that further quality control studies are conducted on a larger scale and repeated more frequently.

Enantioseparation of chiral tropa alkaloids by means of cyclodextrin-modified microemulsion electrokinetic chromatography.

CD-modified microemulsion EKC as a CE technique has been applied to the chiral separation of atropine, scopolamine, ipratropium and homatropine. Enantioseparations of these tropa alkaloids were optimized by using a standard oil-in-water (O/W) microemulsion and varying the nature and concentration of CD additives as well as of the organic modifier (methanol, 2-propanol or ACN) whilst keeping the applied voltage of 15 kV and capillary temperature of 30 degrees C constant. The standard (O/W) microemulsion BGE solution consisted of 0.8% w/w octane, 6.6% w/w 1-butanol, 2.0% w/w SDS and 90.6% w/w 10 mM sodium tetraborate buffer (pH 9.2). Enantioseparations with high resolution and short migration times of all tropa alkaloids were achieved by using heptakis(2,3-di-O-methyl-6-O-sulfo)-beta-CD and sulfated beta-CD in the microemulsion BGE and were superior to corresponding CD-modified CE methods.

Impurity profiling of atropine sulfate by microemulsion electrokinetic chromatography.

An oil-in-water microemulsion electrokinetic chromatography (MEEKC) method has been developed and validated for the determination of atropine, its major degradation products (tropic acid, apoatropine and atropic acid) and related substances from plants material (noratropine, 6-hydroxyhyoscyamine, 7-hydroxyhyoscyamine, hyoscine and littorine). Separation of atropine and all impurities was optimized by varying the voltage, the nature of the oil droplet and the buffer, as well as the organic modifier (methanol, 2-propanol or acetonitrile) and the surfactant type and concentration. The optimum O/W microemulsion background electrolyte (BGE) solution consists of 0.8% (w/w) octane, 6.62% (w/w) 1-butanol, 2.0% (w/w) 2-propanol, 4.44% (w/w) SDS and 86.14% (w/w) 10 mM sodium tetraborate buffer pH 9.2. In order to shorten the analysis time a voltage gradient was applied. The validation was performed with respect to specificity, linearity, range, limit of quantification and detection, precision, accuracy and robustness. The established method allowed the detection and determination of atropine sulfate related substances at impurity levels given in the European Pharmacopoeia. Good agreement was obtained between the established MEEKC method and the traditional RP-HPLC method.

Why not using capillary electrophoresis in drug analysis?

CE and related methods are well-established techniques in the analysis of biomolecules, such as DNA and proteins. Even though CE is a rather good alternative to HPLC for the evaluation of the impurity profile and the enantiomeric purity of a drug, it is rarely applied. This might be due to the reservation of national licensing authorities and the pharmacopoeia commissions for several reasons. In this review containing some experimental data we report on several drug examples which demonstrate the superiority of CE over HPLC in special cases, i.e., in the analysis of antibiotics, amino acids and peptides, and the determination of enantiomeric purity. However, in order to make the CE techniques more suitable for pharmacopoeial purposes the general methods describing separation methods have to be complemented with the adjustment of the electrophoretic conditions being necessary to satisfy the system suitability criteria without fundamentally modifying the methods. Taken together CE should be more often applied in drug quality control.

Interlaboratory study of a NACE method for the determination of R-timolol content in S-timolol maleate: assessment of uncertainty.

Analyses of statistical variance were applied to evaluate the precision and practicality of a CD-based NACE assay for R-timolol after enantiomeric separation of R- and S-timolol. Data were collected in an interlaboratory study by 11 participating laboratories located in Europe and North America. General qualitative method performance was examined using suitability descriptors (i.e. resolution, selectivity, migration times and S/N), while precision was determined by quantification of variances in the determination of R-timolol at four different impurity levels in S-timolol maleate samples. The interlaboratory trials were designed in accordance with the ISO guideline 5725-2. This allowed estimating for each sample, the different variances, i.e. between-laboratory (s2(Laboratories)), between-day (s2(Days)) and between-replicate (s2(Replicates)). The variances of repeatability (s2r) and reproducibility (s2R) were then calculated. The estimated uncertainty, derived from the precision estimates, seems to be concentration-dependent above a given threshold. This example of R-timolol illustrates how a laboratory can evaluate uncertainty in general.

Comparison of the separation of aziridine isomers applying heptakis(2,3-di-O-methyl-6-sulfato)beta-CD and heptakis(2,3-di-O-acetyl-6-sulfato)beta-CD in aqueous and nonaqueous systems.

Aziridines are attracting interest as protease inhibitors, which might be used, e.g., for treatment of parasitic diseases. Within the framework of greater projects dealing with the search of new selective protease inhibitors, a huge number of aziridines with two stereogenic centers will be synthesized. Thus, a fast and reliable screening method for the evaluation of the isomeric composition is needed. Robust baseline separations were obtained using heptakis(2,3-di-O-acetyl-6-sulfato)beta-CD (HDAS) in acidic methanol and sulfated beta-CD in acidic phosphate buffer. With HDAS the resolutions were higher and migration times shorter. Thus, the method will be used as a screening method for further isomeric mixtures of aziridines.

Development and validation of a separation method for the diastereomers and enantiomers of aziridine-type protease inhibitors.

Aziridine derivatives are attracting pharmacological interest as protease inhibitors. Due to their two centers of chirality, the aziridines studied here are mixtures of two diastereomers and corresponding enantiomers. Applying cyclodextrin-modified capillary electrophoresis resulted in a baseline separation of the four isomers. The most robust separation was obtained by means of 2 mM sulfated beta-cyclodextrin in 50 mM phosphate buffer of pH 2.5. Using this method, 0.25% of the trans-diastereomers aziridine could be precisely and accurately quantified in the presence of 99.75% of the cis-isomers. The corrected peak-area ratios, migration times, and resolutions were found to be robust with respect to small variations of voltage, buffer concentrations, pH, temperature, chiral selector concentration, and different lots.