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.

Ionisable substances chromatography: A new approach for the determination of Ketoprofen, Etoricoxib, and Diclofenac sodium in pharmaceuticals using ion - pair HPLC.

An ion-pair HPLC method was developed and validated to analyze three of non-steroidal anti-inflammatory drugs (Ketoprofen, Etoricoxib, and Diclofenac sodium) in their pure and pharmaceuticals based on their ionisable characteristics. Cetyltrimethylammonium bromide (Cetrimide) was used as an ion pair reagent since it had not been used before for this purpose. Chromatographic analysis was accomplished using the C18 (250 × 4.6 mm, 5µm) column. Mobile phase consisted of a mixture of 50% Cetrimide 10 - 3 M and 50% acetonitrile to analyze Ketoprofen and Etoricoxib, whereas for Diclofenac sodium, mobile phase was a mixture of 30% Cetrimide 10 - 3 M and 70% acetonitrile. pH value was adjusted if necessary to 10 with ammonium hydroxide. The flow rate was 1mL/min and detection wavelengths were at 254 nm, 234 nm, and 254 nm for Ketoprofen, Etoricoxib, and Diclofenac sodium; respectively under ambient temperature. Retention times ( R t ) were 9.41, 7.34, and 6.66 for Ketoprofen, Etoricoxib, and Diclofenac sodium; respectively. The proposed method was evaluated for linearity, accuracy, precision, and specificity according to ICH guidelines. Ketoprofen, Etoricoxib, and Diclofenac sodium were detected in the following linear ranges: (0.031-0.500mg/mL), (0.007-0.110g/mL), and (0.016-0.250mg/mL); respectively with excellent mean recovery values (98.0-102.0%). RSD% was in an acceptable range (less than 2), proving the precision of the developed method. Specificity was proved in the presence of degradation products. Furthermore, a comparison between the results of this study and the reported HPLC methods indicated that this developed method was better in terms of simplicity, analysis time, and no use of buffers in the mobile phase. In conclusion, the developed method can successfully detect Ketoprofen, Etoricoxib, and Diclofenac sodium quantitatively and qualitatively in their dosage forms without any interference with excipients, making this method valuable, reliable, and practical to be applied in quality control laboratories.

The usefulness of 2D DOSY and 3D DOSY-COSY 1H NMR for mixture analysis: application to genuine and fake formulations of sildenafil (Viagra).

Two-dimensional diffusion ordered spectroscopy (DOSY) (1)H NMR is proposed to analyze drugs that are complex mixtures in order to discriminate genuine from fake formulations. The method was applied to the analysis of 17 formulations of sildenafil, one being genuine Viagra and the others illegally manufactured formulations of this drug coming from India, Syria and China. It enabled (i) distinguishing imitations or counterfeit from the authentic formulation, (ii) detecting the presence of sildenafil or adulterants, (iii) gaining information on the formulation process by detection of various excipients, thus giving a precise and global 'signature' of the manufacturer. Even though some samples are slightly overdosed, the quality of products manufactured in India and Syria was better than that of Chinese formulations which were adulterated with vardenafil and homosildenafil. This study also presents a three-dimensional DOSY-COSY (1)H NMR experiment that provides both virtual separation and structural information.

2D and 3D DOSY 1H NMR, a useful tool for analysis of complex mixtures: application to herbal drugs or dietary supplements for erectile dysfunction.

Seventeen herbal dietary supplements, marketed as natural substances for the enhancement of sexual function, were analyzed by diffusion ordered spectroscopy (DOSY) (1)H NMR. The method allowed a global analysis of the samples with detection of both active and inactive ingredients present in these complex matrixes. Eight formulations contained compounds related to the synthetic phosphodiesterase-5 inhibitors. Sildenafil, tadalafil, vardenafil, hydroxyhomosildenafil, thiosildenafil, and the newly identified adulterant thiomethisosildenafil were detected. Quantification of these active ingredients was carried out by HPLC or NMR. In addition to these actives, about 30 compounds or excipients were characterized. This study ended up with a three-dimensional DOSY-COSY (1)H NMR experiment on a herbal formulation which provided both virtual separation and structural information.

Analysis of illegally manufactured formulations of tadalafil (Cialis) by 1H NMR, 2D DOSY 1H NMR and Raman spectroscopy.

Counterfeit and/or imitation medicines are becoming a major health problem not only in developing countries but also in wealthier countries. The need of new and easy analytical methods for quality control of drugs is essential. We describe the use of Raman spectroscopy, 1H nuclear magnetic resonance (NMR) and 2D diffusion-ordered spectroscopy (DOSY) NMR to analyse genuine Cialis and seven illegally manufactured formulations of this drug purchased via the internet. Seven out of the eight commercial formulations of tadalafil contain the active ingredient, measured by high performance liquid chromatography (HPLC), within 100+/-5% of stated concentration. Vardenafil and homosildenafil instead of tadalafil were found in the Chinese imitation. 2D DOSY NMR spectra clearly showed similarities and differences in the composition of the pharmaceutical formulations of tadalafil, thus giving a precise and global "signature" of the manufacturer. Our data show that the quality of the Cialis imitations manufactured in India and Syria is correct, whereas the Chinese formulation is adulterated with active pharmaceutical ingredients.

Quality assessment of fluoxetine and fluvoxamine pharmaceutical formulations purchased in different countries or via the Internet by 19F and 2D DOSY 1H NMR.

A simple and selective (19)F NMR method has been validated for the quantitation of fluoxetine (FLX) and fluvoxamine (FLV) in methanol solutions and in human plasma and urine. The regression equations for FLX and FLV showed a good linearity in the range of 1.4-620 microg mL(-1) (3.3 x 10(-6)-1.8 x 10(-3) mol L(-1)) with a limit of detection of approximately 0.5 microg mL(-1) (1.3 x 10(-6) mol L(-1)) and a limit of quantification of approximately 2 microg mL(-1) (4.6 x 10(-6) mol L(-1)). The precision of the assay depends on the concentrations and is comprised between 1.5 and 9.5% for a range of concentrations between 1.2 x 10(-3) and 3.2 x 10(-6) mol L(-1). The accuracy evaluated through recovery studies ranged from approximately 96 to 103% in methanol solutions and in urine, and from approximately 93 to 104% in plasma, with standard deviations <7.5%. The low sensitivity of the method precludes its use for the assay of these antidepressants in biofluids at least at therapeutic doses as the ranges of FLX and FLV plasma levels are 0.15-0.5 microg mL(-1) and 0.15-0.25 microg mL(-1), respectively. The method was applied successfully to the determination of FLX and FLV contents in pharmaceutical samples (brand-named and generic) purchased in several countries or via the Internet. All the commercial formulations contain the active ingredient in the range 94-103% of stated concentration. A "signature" of the formulations (solid and liquid) was obtained with 2D Diffusion-Ordered SpectroscopY (DOSY) (1)H NMR which allowed the characterisation of the active ingredient and excipients present in the formulations studied. Finally, the DOSY separation of FLX and FLV whose molecular weights are very close was obtained by using beta-cyclodextrin as host-guest complexing agent.

Generic ciprofloxacin tablets contain the stated amount of drug and different impurity profiles: A 19F, 1H and DOSY NMR analysis.

The objective of this study was to control the purity of 16 commercial formulations of ciprofloxacin tablets purchased in different countries or via the Internet using 19F and 1H nuclear magnetic resonance (NMR). Twelve out of the sixteen commercial formulations of ciprofloxacin measured by 19F NMR contain the active ingredient within 100+/-5% of stated concentration. Three formulations have a lower ciprofloxacin content between 90 and 95% and one shows a higher concentration superior to 105%. The impurity profile was characterised using 19F and 1H NMR, and is characteristic of the manufacturer. Four to twelve fluorinated impurities among them fluoride ion and two already known compounds were detected and quantified in the sixteen formulations analysed by 19F NMR. Two other non-fluorinated impurities were observed in the seven formulations analysed with 1H NMR. The total content of impurities as well as their individual levels are in agreement with those reported previously in the few studies devoted to ciprofloxacin purity. However, all the formulations do not comply with the limits for impurities given in the ciprofloxacin monograph of the European Pharmacopeia. Finally, a "signature" of the formulations was obtained with Diffusion-Ordered SpectroscopY (DOSY) 1H NMR which allowed the characterisation of some excipients present in the formulations studied.