Development and Validation of a Rapid High-Performance Liquid Chromatography⁻Tandem Mass Spectrometric Method for Determination of Folic Acid in Human Plasma.

There are health concerns associated with increased folic acid intake from fortified food and supplements. Existing analytical methods, however, which can be employed to carry out epidemiological and bioavailability studies for folic acid involve laborious sample preparation and/or lengthy chromatographic analysis. In this paper we describe a simple, rapid, and sensitive high-performance liquid chromatography⁻electrospray ionisation-tandem mass spectrometry (HPLC⁻ESI-MS/MS) method for determination of unmetabolised folic acid in human plasma using folic acid-d4 as an internal standard. The method required only a simple sample preparation step of protein precipitation and had a total run time of 3.5 min, which is the shortest run time reported to date for HPLC⁻MS/MS method employed for quantifying folic acid in plasma. The analytes were separated on a C18 column (3 µm; 50 × 3.00 mm) using an isocratic mobile phase consisting of ammonium acetate (1 mM)-acetic acid-acetonitrile (9.9:0.1:90, v/v/v). The method was fully validated in terms of accuracy, precision, linearity, selectivity, recovery, matrix effect, and stability. The short run time and the minimal sample preparation makes the method a valuable tool for performing high-throughput analyses. To demonstrate the applicability of the method in real conditions, it was applied successfully in a bioavailability study for the determination of unmetabolised folic acid levels in vivo in human plasma after oral administration of folic acid.

Formulation and In Vitro, In Vivo Correlation Between Two Candesartan Cilexetil Tablets.

In this study, the in vitro and in vivo interchangeability between generic candesartan 16 mg and the branded formulation was assessed. The in vitro release of these products was conducted in 3 pH media (1.2, 5.0, and 6.8), and similarity factors (f2 ) were calculated. This bioequivalence study was a randomized, 2-period crossover study that included 42 healthy adult male subjects under fasting conditions with a 9-day washout. The pharmacokinetic (PK) parameters AUC0-last , AUC0-∞ , and Cmax , tmax , and the elimination half-life time were assessed based on the plasma concentrations of candesartan, using a newly developed and validated liquid chromatography-tandem mass spectrometry bioanalytical method with acceptable degrees of linearity, sensitivity, precision, and accuracy. The geometric mean (ng·h/mL) of the AUC0-∞ for the test and brand was 1595.49 and 1620.54, respectively, and the Cmax (ng/mL) was 160.91 and 160.88, respectively. The 90%CIs of geometric mean ratios (test-to-reference ratios) were 98.26%, 98.45%, and 99.86% for AUC0-last , AUC0-∞ , and Cmax respectively. These PK parameters lie within the US Food and Drug Administration- and European Medicines Agency-specified bioequivalence limit (80%-125%). Both products were well tolerated by all the subjects. The tested drug product was bioequivalent to the reference drug and had the same safety profile.

Lornoxicam Immediate-Release Tablets: Formulation and Bioequivalence Study in Healthy Mediterranean Volunteers Using a Validated LC-MS/MS Method.

This study aimed to demonstrate interchangeability between 2 lornoxicam tablet formulations under fasting conditions among Mediterranean Arabs by using a newly validated high-pressure liquid chromatography-tandem mass spectrometry method. A single-oral solid dosage form (8 mg/tablet), randomized, open-label, 2-way crossover study was conducted on 30 healthy male volunteers. Blood samples were collected prior to dosing and over a 24-hour period, and the washout period was 9 days. Statistical comparison of the main pharmacokinetic parameters showed no significant difference between generic and branded products. The point estimates (ratios of geometric mean %) were 90.91, 96.34, and 94.86 for Cmax, AUC0-last , and AUC0-∞ , respectively. The 90% confidence intervals were within the predefined limits of 80.00%-125.00%, as specified by the international guidelines. This study showed that both formulations met the regulatory criteria for bioequivalence.

Using Supercritical Fluid Technology (SFT) in Preparation of Tacrolimus Solid Dispersions.

Tacrolimus is an immunosuppressant agent that suffers from poor and variable bioavailability. This can be related to limited solubility and dissolution. The main objective of this study is to use SFT to prepare solid dispersions of tacrolimus in order to enhance its dissolution. SFT was selected since it offers several advantages over conventional techniques such as efficiency and stability. Several solid dispersions of tacrolimus were prepared using SFT to enhance its dissolution. The selected polymers included soluplus, PVP, HPMC, and porous chitosan. TPGS was used as a surfactant additive with chitosan, HPMC, and PVP. Soluplus dispersions were used to study the effect of processing parameters (time, temperature, and pressure) on loading efficiency (LE) and dissolution of the preparation. Physicochemical characterization was performed using DSC, X-ray diffraction, FTIR analysis, SEM, and in vitro drug release. Stability testing was evaluated after 3 months for selected dispersions. Significant improvement for the release profile was achieved for the prepared dispersions. Better release achieved in the soluplus dispersions which reached maximum cumulative release equal to 98.76% after 24 h. Drug precipitated in its amorphous form in all prepared dispersions except those prepared from chitosan. All dispersions were physically stable except for PVP preparations that contained TPGS which started to re-crystallize after one month. Prepared dispersions were proved to be affected by supercritical processing parameters. In conclusion, SFT was successfully used to prepare dispersions of tacrolimus that exhibited higher dissolution than raw drug. Dissolution rate and stability are affected by the type of the polymer.

Bioequivalence assessment of two pregabalin capsules in healthy Mediterranean Arab volunteers.

BACKGROUND: Treatment of neuropathic pain has always been challenging, not only from the pharmaco-therapeutic/toxicological point of view, but also due to the unpredictable pharmacokinetic (PK) variations among different generic formulations of the same drug, which require further dose optimization. OBJECTIVES: This progressive work aims to evaluate the bioequivalence (BE) of a generic product of 150 mg pregabalin capsule (antineuropathic drug) vs. the reference brand drug Lyrica^®. METHOD: An LC-MS/MS bioanalytical method was developed and validated according to the International Conference on Harmonization (ICH) guidelines in order to be used for the analysis of pregabalin in plasma. BE of capsules was tested by comparison against the reference brand capsules in accordance with the requirements of the declarations of Helsinki, the current Good Clinical Practice (GCP) Guidelines and the ICH. The resulting data were compared against corresponding pregabalin data published on other human races. RESULTS: The relationship between concentration and peak area ratio was found to be linear within the range 0.096 - 6.068 µg/mL for pregabalin. The correlation coefficient (r) was equal to 0.9983. Statistical comparison of the main PK parameters showed no significant difference between test and reference. The mean Cmax values for test and reference were 4.290 and 4.164 µg/mL, and the mean AUC0-last values were 24.275 h×µg/mL and 23.674 h×µg/mL, respectively. The 90% CIs of geometric mean ratios (test/reference) for pregabalin were 100.34 - 104.78%, 100.34 - 104.70%, and 95.65 - 110.96% for AUC0-last, AUC0-∞, and Cmax, respectively, thus fall within the international specified BE limit (80 - 125%). Both products were well tolerated by all the volunteers and there were no significant differences on physical examination or in vital signs and laboratory tests between groups. All volunteers completed the study and were discharged in good health. CONCLUSION: The tested generic capsules appear to be bioequivalent to the reference brand and are expected to have a similar efficacy and safety profile.

Investigation of the Bioequivalence of Rosuvastatin 20 mg Tablets after a Single Oral Administration in Mediterranean Arabs Using a Validated LC-MS/MS Method.

There is a wide inter-individual response to statin therapy including rosuvastatin calcium (RC), and it has been hypothesized that genetic differences may contribute to these variations. In fact, several studies have shown that pharmacokinetic (PK) parameters for RC are affected by race. The aim of this study is to demonstrate the interchangeability between two generic RC 20 mg film-coated tablets under fasting conditions among Mediterranean Arabs and to compare the pharmacokinetic results with Asian and Caucasian subjects from other studies. A single oral RC 20 mg dose, randomized, open-label, two-way crossover design study was conducted in 30 healthy Mediterranean Arab volunteers. Blood samples were collected prior to dosing and over a 72-h period. Concentrations in plasma were quantified using a validated liquid chromatography tandem mass spectrometry method. Twenty-six volunteers completed the study. Statistical comparison of the main PK parameters showed no significant difference between the generic and branded products. The point estimates (ratios of geometric mean %) were 107.73 (96.57-120.17), 103.61 (94.03-114.16), and 104.23 (94.84-114.54) for peak plasma concentration (Cmax), Area Under the Curve (AUC)0→last, and AUC0→∞, respectively. The 90% confidence intervals were within the pre-defined limits of 80%-125% as specified by the Food and Drug Administration and European Medicines Agency for bioequivalence studies. Both formulations were well-tolerated and no serious adverse events were reported. The PK results (AUC0→last and Cmax) were close to those of the Caucasian subjects. This study showed that the test and reference products met the regulatory criteria for bioequivalence following a 20 mg oral dose of RC under fasting conditions. Both formulations also showed comparable safety results. The PK results of the test and reference in the study subjects fall within the acceptable interval of 80%-125% and they were very close to the results among Caucasians. These PK results may be useful in order to determine the suitable RC dose among Arab Mediterranean patients.

Interchangeability and comparative effectiveness between generic and brand montelukast immediate release tablets after a single oral administration in healthy volunteers.

BACKGROUND: Montelukast is a leukotriene receptor antagonist. The release of leukotrienes causes narrowing and constricting in the respiratory airways. Blocking the action of these leukotrienes, montelukast can be used for the prophylaxis and treatment of chronic asthma. OBJECTIVE: The aim of this study was to evaluate the interchangeability and comparative effectiveness between a generic and a brand montelukast 10 mg immediate release tablets (Broncast(®) and Singulair(®), respectively) after a single oral dose among Arab Mediterranean volunteers. METHODS: An open-label, randomized two-period crossover bioequivalence design was conducted in 31 healthy male volunteers with a 1 week washout between each study period and under fasting conditions. The plasma drug concentration was assessed by using a previously validated LC MS/MS method. The ratio between the generic and brand of geometric least squares means was reported for both generic and brand products. Moreover, an in vitro dissolution study was conducted on generic and brand tablets using three different pH media, and similarity and non-similarity factors (f2 and f1) were calculated. RESULTS: The used bioanalytical method was found to be linear within the range 6.098-365.855 ng/mL. The correlation coefficient was close to 0.999 during the course of the study validation. Statistical comparison of the main pharmacokinetic parameters showed the inexistence of any significant difference between generic and the brand. The point estimates (ratios of geometric means) were 111.939, 111.711, and 112.169 % for AUC0-24, AUC0-∞, and Cmax, respectively. The 90 % confidence intervals (CIs) were within the pre-defined limits of 80.00-125.00 % as specified by the FDA and EMA for bioequivalence studies. F2 and f1 were higher than 50 and lower than 15, respectively in all selected pH media. CONCLUSION: Broncast(®) immediate release film coated tablets (10 mg/tablet) are bioequivalent to Singulair(®) immediate release film coated tablets (10 mg/tablet), with a comparable safety and efficacy profile. This suggests that these two formulations can be clinically considered interchangeable. The dissolution study suggests that it could be used as premarketing quality control parameter in order to maintain the high quality of the produced product.

Investigation of the bioequivalence of montelukast chewable tablets after a single oral administration using a validated LC-MS/MS method.

BACKGROUND: Montelukast (MT) is a leukotriene D4 antagonist. It is an effective and safe medicine for the prophylaxis and treatment of chronic asthma. It is also used to prevent acute exercise-induced bronchoconstriction and as a symptomatic relief of seasonal allergic rhinitis and perennial allergic rhinitis. OBJECTIVE: The aim of this study was to evaluate the bioequivalence (BE) of two drug products: generic MT 5 mg chewable tablets versus the branded drug Singulair(®) pediatric 5 mg chewable tablets among Mediterranean volunteers. METHODS: An open-label, randomized two-period crossover BE design was conducted in 32 healthy male volunteers with a 9-day washout period between doses and under fasting conditions. The drug concentrations in plasma were quantified by using a newly developed and fully validated liquid chromatography tandem mass spectrometry method, and the pharmacokinetic parameters were calculated using a non-compartmental model. The ratio for generic/branded tablets using geometric least squares means was calculated for both the MT products. RESULTS: The relationship between concentration and peak area ratio was found to be linear within the range 6.098-365.855 ng/mL. The correlation coefficient (R (2)) was always greater than 0.99 during the course of the validation. Statistical comparison of the main pharmacokinetic parameters showed no significant difference between the generic and branded products. The point estimates (ratios of geometric means) were 101.2%, 101.6%, and 98.11% for area under the curve (AUC)0→last, AUC0→inf, and C max, respectively. The 90% confidence intervals were within the predefined limits of 80.00%-125.00% as specified by the US Food and Drug Administration and European Medicines Agency for BE studies. CONCLUSION: Broncast(®) pediatric chewable tablets (5 mg/tablet) are bioequivalent to Singulair(®) pediatric chewable tablets (5 mg/tablet), with a similar safety profile. This suggests that these two formulations can be considered interchangeable in clinical practice.

Formulation and bioequivalence of two sildenafil 50 mg film-coated tablets after a single oral administration.

OBJECTIVE: The aims of this study were to establish a bioanalytical method and to evaluate the bioequivalence of two drug products; a generic sildenafil 50 mg film-coated vs. the brand drug Viagra® 50 mg film-coated tablets. METHOD: Bioequivalence of tablets was tested by comparisons against the reference brand product in accordance with the requirements of the Declaration of Helsinki, the current Good Clinical Practice (GCP) Guidelines and the International Conference Harmonization (ICH). RESULTS: The relationship between concentration and peak area ratio was found to be linear within the range of 1.435 - 410.023 ng/mL for sildenafil. The correlation coefficient (r) was always greater than 0.99 during the course of the validation Statistical comparison of the main pharmacokinetic parameters showed no significant difference between test and reference. The 90% CIs of geometric mean ratios (test to reference ratios) were 99.656%, 99.806%, and 109.227% for AUC(0-last), AUC(0-∞), and C(max), respectively. These pharmacokinetic parameter values lie within the FDA and European Medicines Agency specified bioequivalence limit (80 - 125%). Both formulations were well tolerated by all subjects and they were discharged in good health. CONCLUSION: The tested drug product was bioequivalent to the reference drug and had the same safety profile.

Comparative fasting bioavailability of two clopidogrel formulations in healthy Mediterranean volunteers: an in vitro-in vivo correlation.

OBJECTIVE: The aim of this study was to evaluate the bioequivalence of two drug products, generic clopidogrel bisulfate 75 mg film-coated tablets versus the reference Plavix(®) clopidogrel bisulfate 75 mg film-coated tablets. METHODS: Bioequivalence of tablets was tested by comparisons against the reference brand product in accordance with the requirements of the Declaration of Helsinki, the current Good Clinical Practice Guidelines, and the International Conference on Harmonization. RESULTS: The relationship between concentration and peak area ratio was found to be linear within the range 24.500-1,836.600 pg/mL for clopidogrel. The correlation coefficient (r) was always greater than 0.99 during the course of the validation. Statistical comparison of the main pharmacokinetic parameters showed no significant difference between test and reference. The point estimates (ratios of geometric mean) were 104.122%, 104.184%, and 109.091% for areas under the plasma concentration-time curve (AUC) AUC0-last, AUC0-∞, and peak plasma concentration C max, respectively. These pharmacokinetic parameter values of clopidogrel and its main metabolite lie within the bioequivalence limit (80%-125%) specified by the US Food and Drug Administration and the European Medicines Agency. CONCLUSION: The tested drug product was bioequivalent to the reference drug under fasting conditions and had the same safety profile, which is important to achieve equivalent therapeutic effect with the reference.

Drying Using Supercritical Fluid Technology as a Potential Method for Preparation of Chitosan Aerogel Microparticles.

Supercritical fluid technology offers several advantages in preparation of microparticles. These include uniformity in particle size, morphology, and drug distribution without degradation of the product. One of the recent advantages is preparation of porous aerogel carrier with proper aerodynamic properties. In this study, we aimed to prepare chitosan aerogel microparticles using supercritical fluid (SCF) technology and compare that with microparticles produced by freeze drying (FD). Loading the prepared carriers with a model drug (salbutamol) was also performed. Comparisons of the particle properties and physicochemical characterizations were undertaken by evaluating particle size, density, specific surface area, and porosity. In vitro drug release studies were also investigated. The effect of many variables, such as molecular weight of chitosan oligomers, concentrations of chitosan, and concentrations of tripolyphosphate on the release, were also investigated. Chitosan aerogels were efficiently produced by SCF technology with an average particle size of 10 µm with a tapped density values around 0.12 g/mL, specific surface area (73-103) m(2)/g, and porosity (0.20-0.29) cc/g. Whereas, microparticles produced by FD method were characterized as cryogels with larger particle size (64 microns) with clear cracking at the surface. Sustained release profile was achieved for all prepared microparticles of salbutamol produced by the aforementioned methods as compared with pure drug. The results also demonstrates that chitosan molecular weight, polymer concentration, and tripolyphosphate concentration affected the release profile of salbutamol from the prepared microparticles. In conclusion, SCF technology was able to produce chitosan aerogel microparticles loaded with salbutamol that could be suitable for pulmonary drug delivery system.

Generation of fine powders of recombinant human deoxyribonuclease using the aerosol solvent extraction system.

PURPOSE: To investigate the feasibility of using the Aerosol Solvent Extraction System (ASES) to produce fine powders of recombinant human deoxyribonuclease (rhDNase), lysozyme-lactose and rhDNase-lactose powders from aqueous based solutions. METHODS: The ASES technique using high pressure carbon dioxide modified with ethanol or ethanol and triethylamine was used for the generation of rhDNase powders and protein-lactose powders from aqueous based solutions. Particle size, morphology, size distributions, crystallinity, and powder aerosol performance were measured. The biochemical integrity of the processed rhDNase was assessed by testing the monomer content and the degree of deamidation. RESULTS: RhDNase precipitated as spherical particles in the size range between 50 and 500 nm. The primary nano-sized particles were agglomerated to micron-sized clumps of particles during the precipitation process. The median particle size and the fine particle fraction were functions of the operating temperature and the nozzle system used. RhDNase was substantially denatured in the ASES process using carbon dioxide modified with ethanol as anti-solvent. However almost complete recovery of the monomer was achieved using carbon dioxide modified with ethanol-triethylamine as an anti-solvent. Lysozyme-lactose and rhDNase-lactose powders were also precipitated as agglomerated spheres using the ASES process. The powders were amorphous except for those with lactose content higher than 45%. CONCLUSIONS: Micron-sized particles of rhDNase suitable for inhalation delivery were generated from aqueous based solutions using the modified ASES technique. The biochemical integrity of the rhDNase powder is a function of the antisolvent and the operating temperature.

A solid-state NMR study of protein mobility in lyophilized protein-sugar powders.

The molecular mobility of protein in lyophilized lysozyme-sugar systems stored at different relative humidities was studied using solid-state NMR. Relaxation measurements, T(1) of high-frequency (MHz), and T(1rho), of low-frequency (kHz) motions, were performed on lysozyme lyophilized with lactose and trehalose. Molecular aggregation and enzymatic activity of the protein were determined using HPLC and bioassays. An increase in hydration had little effect on the T(1rho) values of pure lysozyme, trehalose, lactose, trehalose-lysozyme, and lysozyme at low lactose concentrations. The T(1) values of pure sugar increased as moisture content increased. The presence of both sugars led to increased T(1) values of the lysozyme but increasing hydration gradually reduced T(1) values. When a larger amount of lactose was lyophilized with lysozyme, longer T(1) (and T(1rho)) values were seen for lactose than for lysozyme. Although longer T(1) values were related to an increase in protein stability, the effect of crystallization and sugar type appeared to be major contributing factors. Trehalose and lactose decreased relaxation rates in the lysozyme-sugar systems while hydration increased relaxation rates that were correlated with changes in aggregation and activity of the protein.