ICH Recommendations Research Articles

Innovative ultra-sensitive spectrofluorimetric method for nanogram detection of doripenem monohydrate in human plasma, urine and pharmaceutical formulation

Doripenem monohydrate (DOR), is a recently FDA approved β- lactam antibiotic. DOR belongs to carbapenems class and it is used for the treatment of several diseases caused by different strains of bacteria. Herein, new highly sensitive, low casting, and selective spectrofluorimetric method was developed and validated for determination of DOR in real human plasma. The developed approach based on the reaction of 4-Chloro-7-nitrobenzofurazan (NBD-Cl) with secondary amine moiety in DOR using 0.1 M borate buffer at pH 8.5 producing fluorescent product measured at 535 nm (λex at 475 nm) after heating for 8 min at 70 °C. The calibration range was studied under optimum conditions and it was linear from 30 to 500 ng mL−1, with LOD 9.73 ng mL−1 and LOQ 24.05 ng mL−1. The investigated method was validated and bio-analytically validated using ICH and FDA recommendations. Moreover, the proposed method was successfully utilized to quantify DOR in a commercial formulation, human plasma and urine with high percentage of recovery in the range of 97.26% to 98.89% for plasma samples and within the range of 99.28% to 99.84% for urine samples. Further, the proposed method was successfully applied for extensive pharmacokinetic study in order to improve patient health.

Utility of fluorescamine-based approach for highly sensitive spectrofluorimetric determination of Ceftazidime and Vancomycin in pharmaceuticals and real human plasma

Ceftazidime pentahydrate (CEF) and Vancomycin hydrochloride (VAN) are antimicrobial drugs they are used worldwide especially in developing countries for management several diseases caused by bacterial infections especially against Pseudomonas species. From this point, ultrasensitive, simple, rapid, cost effective method was developed for assay of CEF and VAN in real human plasma. The proposed spectrofluorimetric method was achieved using fluorescamine reagent. This method based on reaction of fluorescamine reagent with primary amine moiety (aromatic and aliphatic) in CEF and VAN respectively, with calibration graph from 30 to 300 ng mL−1 and 0.5–30 ng mL−1 was plotted under optimum conditions. The investigated method was developed and bio-analytically validated using ICH and US-FDA recommendations. The proposed method was successfully used for determination of the cited drugs in real human plasma with high percentage of recovery ranged from 95.72 ± 0.95% to 97.72 ± 2.02 and pharmaceutical formulations with percentage 101.55 ± 0.55% and 101.99 ± 0.43% for CEF and VAN respectively. The study was also utilized to examine the stability of the CEF and VAN in real human plasma.

Development and validation of the method for determination of encapsulation efficiency of cytochrome c in liposomes

A strategic pathway in creating high-potent medical products is with targeted therapeutic systems that are based on nanoparticles of various structure. Such particles are capable of providing a targeted effect and an increase in bioavailability of the medical products. A special place among modern targeted drug delivery systems is held by liposomal nanoparticles which have apparent advantages over nanoparticles of an another type The problem number one for pharmacy lies in developing specific methods of control of nanosize drug delivery systems.
 This research is dedicated to the development and validation of a technique for determining encapsulation efficiency of cytochrome C in liposomes.
 The subject of research comprised the obtained liposomal form of cytochrome C, placebo emulsion, and cytochrome C solution. The research was conducted in compliance with the ICH and FDA requirements and recommendations in relation to the development of HPLC methods of control of liposomal preparations.
 A method has been developed to enable the determination of encapsulation efficiency of cytochrome C in liposomal preparations and to allow for identifying the composition of liposomal nanoparticles of cytochrome C. This HPLC method has been validated in terms of specificity, limit of detection, and robustness in compliance with the recommended criteria. The technique may find its application in quality control of liposomal form of cytochrome C and in control points manufacturing process.

Validated stability-indicating spectrophotometric methods for the determination of Silodosin in the presence of its degradation products

Five simple, rapid, accurate, and precise spectrophotometric methods are developed for the determination of Silodosin (SLD) in the presence of its acid induced and oxidative induced degradation products. Method A is based on dual wavelength (DW) method; two wavelengths are selected at which the absorbance of the oxidative induced degradation product is the same, so wavelengths 352 and 377 nm are used to determine SLD in the presence of its oxidative induced degradation product. Method B depends on induced dual wavelength theory (IDW), which is based on selecting two wavelengths on the zero-order spectrum of SLD where the difference in absorbance between them for the spectrum of acid induced degradation products is not equal to zero so through multiplying by the equality factor, the absorption difference is made to be zero for the acid induced degradation product while it is still significant for SLD. Method C is first derivative (1D) spectrophotometry of SLD and its degradation products. Peak amplitudes are measured at 317 and 357 nm. Method D is ratio difference spectrophotometry (RD) where the drug is determined by the difference in amplitude between two selected wavelengths, at 350 and 277 nm for the ratio spectrum of SLD and its acid induced degradation products while for the ratio spectrum of SLD and its oxidative induced degradation products the difference in amplitude is measured at 345 and 292 nm. Method E depends on measuring peak amplitudes of the first derivative of the ratio (1DD) where peak amplitudes are measured at 330 nm in the presence of the acid induced degradation product and measured by peak to peak technique at 326 and 369 nm in the presence of the oxidative induced degradation product. The proposed methods are validated according to ICH recommendations. The calibration curves for all the proposed methods are linear over a concentration range of 5–70 μg/mL. The selectivity of the proposed methods was tested using different laboratory prepared mixtures of SLD with either its acid induced or oxidative induced degradation products showing specificity of SLD with accepted recovery values. The proposed methods have been successfully applied to the analysis of SLD in pharmaceutical dosage forms without interference from additives.

Preparation and characterization of in situ carbon paste and screen-printed potentiometric sensors for determination of econazole nitrate: surface analysis using SEM and EDX

The construction and performance characteristics of new sensitive and selective in situ carbon paste (ICPE) and screen-printed (ISPE) potentiometric sensors modified with ion-pairing agents such as phosphotungstic acid, sodium tetraphenylborate, phosphomolybdic acid and ammonium reineckate for determination of econazole nitrate (ECN) have been developed. The reaction mechanism between ECN and ion-pairing agents at the electrode surface was studied through scanning electron microscope and energy-dispersive X-ray analysis. The electrodes under investigation showed potentiometric response for ECN in the concentration range from 1.0 × 10−6 to 5.0 × 10−3 mol L−1 and from 1.0 × 10−6 to 1.0 × 10−2 mol L−1 for ISPE (electrode I) and ICPE (electrode II) potentiometric sensors, respectively, at 25 °C. The electrode response was pH independent in the range 2.5–7.5 and 2.5–6.5 for electrodes I and II, respectively. These sensors have Nernstian slope values of 59.4 ± 0.2 and 59.10 ± 0.2 mV decade−1 with detection limit of 1.0 × 10−6 mol L−1 for electrodes I and II, respectively. The electrodes showed fast response time of 4 and 9 s for electrodes I and II, respectively. The ISPE (electrode I) showed lifetime of 28 days, and this was considered as advantage over ICPE (electrode II). Selectivity for ECN with respect to a number of interfering materials was also investigated. The proposed electrodes were applied for determination of ECN in pure and pharmaceutical formulation using calibration, potentiometric titration and standard addition methods. The results showed good agreement with those obtained using official method. The t and F values indicated no significant difference between the suggested and reported methods. Method validation parameters were optimized according to ICH recommendations.

Occurrence of biogenic amines in beers from Chilean market

The presence of tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine was determined for the first time in beers commercialized in Chilean market. Dansylated amines were separated on a Zorbax-XDB C18 column using a binary mobile phase composed of acetonitrile and 20 mmol L−1 ammonium formate, pH 5.5. Chromatographic conditions were optimized using an experimental design, and validation was carried out following ICH recommendations. Calibration data fitted a linear regression model with R2 > 0.996. Repeatability (n = 6) and intermediate precision (n = 3) in matrix showed RSD values lower than 5.00% and 5.21%, respectively. Recoveries at three different concentrations ranged from 75.50 to 96.48%. The proposed method was applied to determine the biogenic amines content in 101 beer samples produced by macro- (n = 65) and microbreweries (n = 36) using low (n = 63) and high fermentations (n = 38). Biogenic amines content in beer samples ranged from 0.53 to 85.04 mg L−1, from which beers produced by microbreweries (19.13 ± 16.92 mg L−1) showed a significant higher biogenic amines content (P = 0.0021) than beers produced by macrobreweries (9.65 ± 4.50 mg L−1). Putrescine was the principal biogenic amine found in all kind of beers regardless origin, and the type of fermentation and brewery. Only 2 samples presented relevant levels of histamine and tyramine, but both below the limits reported by the European Food Safety Authority (EFSA). Thus, it can be concluded that beers commercialized in Chile are not a serious toxicological risk (e.g. hypertensive crisis) regarding the type and content of biogenic amines.

A validated reverse phase stability-indicating HPLC method for bortezomib in the presence of degradation products and its process-related impurities

Degradation pathway of bortezomib was established as per ICH recommendations in terms of validated and stability indicating reverse phase liquid chromatographic method. Bortezomib was subjected to stress using conditions of acid, base, oxidation, thermal and photolysis. Significant degradation was observed in acid and base stress conditions. Ten impurities were studied and the major degradant was hydroxyamide impurity. The stress samples were assayed against a qualified reference standard and the mass balance is found close to 98.2%. Efficient chromatographic separation was achieved on a Zorbax Extend C18(100 x 4.6 mm, 1.8 μm) stationary phase with simple mobile phase combination In the developed LC method, the resolution between bortezomib and ten potential impurities such as Imp-A, Imp-B, Imp-C, Imp-D, Imp-E, Imp-F, Imp-G, bortezomib isomer, hydroxyamide and bortezomib ester) was found to be greater than 2.0. Regression analysis showed r value (correlation coefficient) of greater than 0.999 for bortezomib and ten potential impurities. This method was capable of detecting the impurities of bortezomib at a level of 0.02% with respect to test concentration of 2.0 mg/mL. The developed rapid LC method was validated with respect to specificity, linearity, range, accuracy, precision and robustness for impurities.

Implementation of Analytical Control of Low Volume Pediatric Cytotoxic Drugs Preparations using a UV/Raman Spectrophotometer

Abstract: Pediatric chemotherapy preparations are usually not analytically controlled, for several reasons. First, they are generally made in syringe, which does not allow to take a sample without changing the final volume. Secondly, the percentage of the dose consecrated to control is important and finally low concentrations can cause sensitivity problems. This lack of quality control, greatly reduces the security of the chemotherapy circuit.: The main objective is to develop an analytical control to low volume pediatric preparations, made in syringes or in infusion bags with a final volume from 20 to 50 mL.: The development of analytical control automatons, like QCPrep+: Our protocol implies the overfilling with one milliliter of solvent followed by a sampling of one milliliter. The analysis is performed with 900 µL. Ten cytotoxic drugs commonly used in pediatric oncology have been validated according to ICH recommendations. The development of analytical control for low volume preparations allows young patients to benefit the same level of safety and quality than adult patients.

Stability Indicating Reverse Phase Chromatographic Method for Estimation of Related Substances in Voriconazole Drug Substance by Ultra Performance Liquid Chromatography

A simple, sensitive and accurate analytical method for related substances of Voriconazole has been developed and validated by using RP-UPLC technique. The developed analytical procedure was validated as per ICH recommendations. Analysis was performed on a HALO C18 (100 × 2.1 2.7 μ) column. 20 mM ammonium formate adjusted the pH to 4.5 with formic acid was selected as buffer. Acetonitrile was used as organic modifier in the mobile phase composition. A simple gradient was applied in the method. Flow rate of mobile phase was kept at 0.4 ml per min. Column compartment temperature was maintained at 45°C. Injection volume was set at 1 μL with an auto sampler maintained the temperature at 10°C. Detection of all the components was monitored at 254 nm by photodiode array detector. Developed method satisfies the system suitability criteria, peak integrity, and resolution for the parent drug and its related substances. The proposed method was validated for Specificity, precision, accuracy, linearity, limit of detection and quantification. Forced degradation studies were conducted to assess stability indicating nature of the method under acidic, basic, oxidative and photolytic conditions. Run time less than 7.0 minutes indicating that the method is cost effective and productive; it can be successfully applied for testing of related substances in drug substance and assay of drug substance in routine quality control analysis and stability testing.

Fluorimetric determination of diosmin and hesperidin in combined dosage forms and in plasma through complex formation with terbium

A sensitive and simple fluorimetric method was developed for the determination of diosmin and hesperidin. The proposed method involves the formation of ternary complex with Tb3+ in the presence of Tris buffer. The fluorescence quenching of Tb3+ at 549 and 494nm (λex at 275 and 248nm) due to the complex formation was quantitatively measured for diosmin and hesperidin, respectively. The reaction conditions and the fluorescence spectral properties of the complexes have been investigated. Under the described conditions, the proposed method was applicable over the concentration range (4.93×10−6–1.81×10−5mol) and (3.28×10−6–1.64×10−5mol) with mean percentage recoveries 100.22±0.89 and 99.13±0.72 for diosmin and hesperidin, respectively. The proposed method was applied successfully for the determination of studied drugs in bulk powder, dosage forms and plasma samples. The results obtained by applying the described method were statistically analyzed and compared with those obtained by applying a reported method. The method was validated according to ICH recommendations.

Degradation Pathway for Eplerenone by Validated Stability Indicating UP-LC Method.

Degradation pathway for eplerenone is established as per ICH recommendations by validated and stability-indicating reverse phase liquid chromatographic method. Eplerenone is subjected to stress conditions of acid, base, oxidation, and thermal and photolysis. Significant degradation is observed in acid and base stress conditions. Four impurities are studied and the major degradant (RRT about 0.31) was identified by LC-MS and spectral analysis. The stress samples are assayed against a qualified reference standard and the mass balance is found close to 99.5%. Efficient chromatographic separation is achieved on a Waters symmetry C18 stationary phase with simple mobile phase combination delivered in gradient mode and quantification is carried at 240 nm at a flow rate of 1.0 mL min−1. In the developed LC method the resolution between eplerenone and four potential impurities (imp-1, imp-2, imp-3, and imp-4) is found to be greater than 4.0. Regression analysis shows an r value (correlation coefficient) of greater than 0.999 for eplerenone and four potential impurities. This method is capable to detect the impurities of eplerenone at a level of 0.020% with respect to test concentration of 1.0 mg mL−1 for a 20 μL injection volume. The developed UPLC method is validated with respect to specificity, linearity and range, accuracy, precision, and robustness for impurities and assay determination.

Degradation Pathway for Rilpinavir Hydrochloride by Validated Stability Indicating UP-LC Method

Degradation pathway for Rilpivirine Hydrochoride is established as per ICH recommendations by validated and stability indicating reverse phase liquid chromatographic method. Rilpivirine Hydrochoride is subjected to stress conditions of acid, base, oxidation, thermal and photolysis. Significant degradation is observed in acid and base stress conditions. Six impurities are studied and the major degradant (RRT about 0.52) is identified by LC-MS and spectral analysis. The stress samples are assayed against a qualified reference standard and the mass balance is found close to 99.5 %. Efficient chromatographic separation is achieved on a Shimpack XR –ODS-II stationary phase with simple mobile phase combination delivered in gradient mode and quantification is carried at 295 nm at a flow rate of 1.0 mL min-1.In the developed LC method the resolution between Rilpivirine Hydrochoride and six potential impurities (imp-1, imp-2, imp-3, imp-4, imp-5 and imp-6) is found to be greater than 2.0. Regression analysis shows an r value (correlation coefficient) of greater than 0.995 for Rilpivirine Hydrochoride and six potential impurities. This method is capable to detect the impurities of Rilpivirine Hydrochoride at a level of 0.006 % with respect to test concentration of 1.0 mg mL-1 for a 4-µL injection volume. The developed LC method is validated with respect to specificity, linearity & range, accuracy, precision and robustness for impurities and degradant determination.

Stability-indicative HPLC determination of donepezil hydrochloride in tablet dosage form

Asimple, selective, precise and stability-indicative high-performance liquid chromatography (HPLC) method of analysis of donepezil hydrochloride in tablet dosage form has been developed and validated. The drug undergoes degradation under acidic, basic, peroxide, photochemical and thermal conditions. The method has been statistically validated for linearity, accuracy, precision, limit of detection, limit of quantitation, solution stability, and selectivity according to ICH recommendations. Due to its simplicity and accuracy, the method can be used for routine drug quality control.

A Validated Stability-Indicating UF LC Method for Bortezomib in the Presence of Degradation Products and its Process-Related Impurities

Degradation pathway for bortezomib is established as per ICH recommendations by validated and stability indicating reverse phase liquid chromatographic method. Bortezomib is subjected to stress conditions of acid, base, oxidation, thermal and photolysis. Significant degradation is observed in acid and base stress conditions. Five impurities are studied and the major degradant (RRT about 1.19) was identified by LC-MS and spectral analysis. The stress samples are assayed against a qualified reference standard and the mass balance is found close to 99.5%. Efficient chromatographic separation is achieved on a Shim pack XR-ODS-II(100X3 mmx2.2 μm) stationary phase with simple mobile phase combination delivered in gradient mode and quantification is carried at 270 nm at a flow rate of 0.6 mL min. In the developed LC method the resolution between bortezomib and five potential impurities (imp-1, imp-2, imp-3, imp-4 and imp-5) is found to be greater than 2.0. Regression analysis shows an r value (correlation coefficient) of greater than 0.999 for bortezomib and five potential impurities. This method is capable to detect the impurities of bortezomib at a level of 0.020 % with respect to test concentration of 2.0 mg mL for an 8-μL injection volume. The developed rapid LC method is validated with respect to specificity, linearity & range, accuracy, precision and robustness for impurities determination.

A Novel Validated Stability Indicative UP-LC Method for Etravirine for the Determination of Process Related and Degradation Impurities

A novel stability indicating reverse phase ultra performance liquid chromatographic (UP-LC) method has been developed for Etravirine along with eight impurities (imp-1, imp-2, imp-3, imp-4, imp-5, imp-6, imp-7 and imp-8) and validated as per ICH recommendations. Stress degradation conditions were established for Etravirine by subjecting it to stress conditions of acid, base, oxidation, humidity, thermal and photolysis. Significant degradation is observed in base stress condition and the major degradant (RRT at about 0.94) is identified by LC-MS and spectral analysis. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.0%. Efficient chromatographic separation was achieved on a Shimpack ODS-II stationary phase with a gradient mobile phase combination. Quantification was carried at 303 nm at a flow rate of 0.6 mL?min–1. The resolution between Etravirine and eight potential impurities is found to be greater than 2.0. Regression analysis shows as r value (correlation coefficient) of greater than 0.999 for Etravirine and eight potential impurities. This method is capable to detect the impurities of Etravirine at a level of 0.003% with respect to test concentration of 1.0 mg·mL–1.

Identification of Degradant Impurity in Gefitinib by Using Validated RRLC Method

Degradation pathway for gefitinib is established as per ICH recommendations by validated and stability in-dicating reverse phase liquid chromatographic method. Gefitinib is subjected to stress conditions of acid, base, oxidation, thermal and photolysis. Significant degradation is observed in acid and base stress condi-tions. Two impurities are studied among which one impurity is found prominent degradant. The stress sam-ples are assayed against a qualified reference standard and the mass balance is found close to 99.5%. Effi-cient chromatographic separation is achieved on a Agilent make XDB-C18, 50 × 4.6 mm with 1.8 µm parti-cles stationary phase with simple mobile phase combination delivered in gradient mode and quantification is carried at 250 nm at a flow rate of 0.5 mL?min-1. In the developed RPLC method the resolution between ge-fitinib and the potential impurities is found to be greater than 5.0. Regression analysis shows an r value (cor-relation coefficient) of greater than 0.998 for gefitinib and the two potential impurities. This method is capa-ble to detect the impurities of gefitinib at a level of 0.01% with respect to test concentration of 0.5 mg?mL-1 for a 4-µL injection volume. The developed RRLC method is validated with respect to specificity, linearity & range, accuracy, precision and robustness for impurities determination and assay determination.

Degradation Pathway for Pitavastatin Calcium by Validated Stability Indicating UPLC Method

Degradation pathway for pitavastatin calcium is established as per ICH recommendations by validated and stability indicating reverse phase liquid chromatographic method. Pitavastatin is subjected to stress conditions of acid, base, oxidation, thermal and photolysis. Significant degradation is observed in acid and base stress conditions. Four impurities are studied among which impurity-4 is found prominent degradant. The stress samples are assayed against a qualified reference standard and the mass balance is found close to 99.5%. Efficient chromatographic separation is achieved on a BEH C18 stationary phase with simple mobile phase combination delivered in gradient mode and quantification is carried at 245 nm at a flow rate of 0.3 mL min-1. In the developed UPLC method the resolution between pitavastatin calcium and four potential impurities is found to be greater than 4.0. Regression analysis shows an r value (correlation coefficient) of greater than 0.998 for pitavastatin calcium and four potential impurities. This method is capable to detect the impurities of pitavastatin calcium at a level of 0.006% with respect to test concentration of 0.10 mg/mL for a 2-µL injection volume. The developed UPLC method is validated with respect to specificity, linearity & range, accuracy, precision and robustness for impurities determination and assay determination.

Isocratic rapid liquid chromatographic method for simultaneous determination of carotenoids, retinol, and tocopherols in human serum

An improved isocratic and rapid HPLC method was developed for the measurement of carotenoids, retinol and tocopherols in human serum. Vitamins were extracted with hexane. Mobile phase consisted of a mixture acetonitrile:methylene chloride:methanol with 20 mM ammonium acetate. This method used a small bead size (3 μm) Spherisorb ODS2 column with titane frits. Diode array and fluorescence detectors were used respectively for the detection of carotenoids and retinol/tocopherols. Chromatographic separation was complete in 13 min for β-cryptoxanthin, cis–trans-lycopene, α-carotene, β-carotene, cis-β-carotene, retinol, δ-tocopherol, γ-tocopherol and α-tocopherol. Echinenone and tocol were employed as internal standards for diode array and fluorescence detection. Accuracy was validated using standard reference material (SRM) 968C. Intra-assay and inter-assay precision were respectively 0.2–7.3% and 3.6–12.6%. Sensitivity was verified using the ICH recommendations and the limit of detection (LOD) obtained was sufficient for routine clinical application.

Pharmaceutical analysis beyond compendial requirements

Recent ICH recommendations on test procedures and acceptance criteria for new drug substances of synthetic chemical origin and new drug products produced from them (Q6A) sets the frame for registration of new chemical entities. These requirements are automatically applied by all pharmaceutical companies to products in late developmental phases. Furthermore, and justifiably so, they do not provide any guideline for setting specifications other than those for residual solvents and residues of heavy metal catalysts. During development of new chemical entities, ICH requirements represent a minimum beyond which additional testing has to be carried out in order to accumulate a body of scientific data which may ultimately permit simplified testing. This information is furthermore needed for drafting of registration files. This presentation will provide information on salt and polymorph selection, as well as morphology of particles. The need for more sophisticated techniques for assay of trace impurities by LC/MS will be illustrated with examples of alkylating agents. Complementary information from techniques such as CE, orthogonal to HPLC, is often needed to ensure absence of additional impurities. Finally, beyond compendial characterisation of hydroxypropylmethylcellulose, a commonly used excipient, by size exclusion chromatography with triple detection will be described.

Enantiomeric purity determination of ketoprofen by capillary electrophoresis: development and validation of the method.

A simple, fast capillary electrophoresis method for determining the total ketoprofen content in an oral pharmaceutical formulation is proposed. The addition of 75 mM of heptakis(tri- O-methyl)-beta-cyclodextrin to the background electrolyte allows the quantitation of the enantiomeric impurity of ( R)-(-)-ketoprofen contained in the formulation. A relative limit of detection is proposed as a measure of the lowest detectable enantiomeric impurity and the results show that the method can detect the minor enantiomer at levels as low as 0.04% in ( S)-(+)-ketoprofen. The chiral method was validated following ICH recommendations and the quality parameters obtained show the suitability of the proposed method. The analysis of samples examined during the course of a stability study under chiral and achiral conditions revealed that the total ketoprofen content did not change significantly with time and the enantiomeric impurity range was 0.1-0.4%.