Precise and Sensitive Ambient Temperature Based Analytical Colorimetric Method for Pregabalin

Abstract Pregabalin, a GABA analogue is used to treat epilepsy and neuropathic pain. The drug poses problems in analytical quantification when estimated at a shorter UV wavelength. The expensive and non-repetitive reported analytical methods necessitate the utility and development of an accurate, precise, repetitive, simple and highly sensitive colorimetric method for pregabalin in solution as well as sustained release mini matrices. Pregabalin (having primary amino group) was derivatized at alkaline pH of mixture with optimized ninhydrin solution at ambient temperature (25oC). The ninhydrin-pregabalin derivatized complex (Ruhemann's Purple) was analyzed for drug concentration at absorption maximum (λmax) of 570nm. The linearity was observed in the concentration range of 5-150 µg/mL with coefficient of correlation, 0.998. The developed analytical method was validated according to ICH guidelines and proved to be highly sensitive (LOD 0.917µg/mL, LOQ 3.055µg/mL), with good inter-day as well as intra-day accuracy and precision as 4.65% and 3.75%, respectively. The proposed method was proved to be a simple, sensitive, precise and accurate for the estimation of the minute concentrations of pregabalin in pure form and the developed formulations. Results verified that the proposed method could determine pregabalin at the ambient temperature without requiring high temperatures used in the existing methods. It was concluded that developed method was easier and more suitable for analysis of pregabalin in quality control of commercial preparations

Comparative application of biological and ninhydrin- derivatized spectrophotometric assays in the evaluation and validation of amikacin sulfate injection

Abstract Instrumental techniques are preferred over bioassay methods for antibiotic quantification mainly due to speed and ability to quantify metabolites in biological samples; however, the potency and biological activity of these drugs cannot be assessed. Two methods - agar well diffusion (bio-assay) and spectrophotometric methods were used to evaluate amikacin sulfate injection. Agar plates were inoculated with S. aureus inoculum; zones of inhibition from its susceptibility to amikacin were obtained, while spectrophotometric absorption at 650 nm of ninhydrin- derivatized amikacin in phosphate buffer (pH 8) was measured. Methods performance showed linearity from 1 - 16 µgmL-1 (bioassay, r = 0.9994) and 10-50 µgmL-1 (spectrophotometric, r = 0.9998). Molar absorptivity was 2.595 x 104 Lmol-1cm-1. Limits of detection and quantification were 1.07 and 3.24 µgmL-1 respectively for bioassay method, while corresponding values for spectrophotometric method were 0.98 and 2.97 µg mL-1. Relative standard deviations were ≤ 2.0% for both methods, with recoveries from 95.93 - 100.25%. Amikacin in brands ranged from 97.53 ± 2.68 to 100.84 ± 1.82%, student's t-test was ≤ 2.78 (n = 4) with respect to label claim for both methods. Experimental paired t-test (t = 2.07; n = 4) and F-test (F = 3.94; n = 4) values indicated no significant difference between both methods, hence comparable and can jointly be used in quality control assessment of antibiotics

Criteria for identification of Schistosoma mansoni eggs in faecal sediments prepared with the Helmintex method and stained by ninhydrin

Helmintex is a sensitive method used for detecting Schistosoma mansoni eggs. Here, we describe the observed frequency of six proposed criteria associated with the identification of S. mansoni eggs prepared with the Helmintex method and stained with ninhydrin. The efficacy of these criteria in classifying S. mansoni eggs when applied in various combinations was also examined. Nine observers registered the presence or absence of 6 different criteria in 100 eggs using a microscope at 100x magnification. Ninhydrin purple, which was frequently observed, was the criterion associated with the lowest inter-observer variability. At least three criteria were associated with a significantly better performance in egg identification. In conclusion, ninhydrin staining and a combination of criteria are recommended for microscope examination of faecal sediments.

Comparação entre três métodos analíticos para determinação de soro em leite cru refrigerado / Comparison of three analytical methods to determine serum in refrigerated raw milk

O objetivo deste experimento foi comparar três métodos analíticos para determinação de soro em leite cru refrigerado: cromatografia líquida de alta eficiência, ninidrina ácida e colorimétrico adaptado. Foram coletadas 100 amostras de leite cru refrigerado de tanques de expansão. Estas, quando submetidas à análise pelo método da ninidrina ácida, apresentaram 10 (14,7%) amostras negativas e 58 (85,3%) positivas. O teor médio de ácido siálico encontrado na técnica da ninidrina foi de 5,58(g/mL, com valor mais frequente de 2,70(g/mL. Das 68 amostras negativas pela cromatografia líquida de alta eficiência, duas foram positivas (2,94%) e 66 (97,06%) negativas, quando analisadas pelo método colorimétrico. A frequência relativa de amostras positivas foi de 32%, com a CLAE apresentando a maior média de soro (14,37%), seguida do método colorimétrico (5,28%) e o da ninidrina ácida (3,12%). A técnica de cromatografia líquida de alta eficiência diferiu dos métodos de ninidrina ácida e colorimétrico, enquanto os métodos da ninidrina e colorimétrico não diferiram entre si, podendo ambos serem utilizados como metodologias de triagem. Entre as três técnicas, a cromatografia líquida de alta eficiência foi a metodologia mais sensível na detecção e quantificação do soro em leite cru refrigerado.(AU)

The objective of this study was to compare three analytical methods to determine serum in refrigerated raw milk. High-performance liquid chromatography (HPLC), acidic and colorimetric ninhydrin methods were applied. A collection of 100 samples of raw milk from cooled expansion tanks took place. The results showed that 10 samples (14.7%) were negative and 58 (85.3%) were positive for the acidic ninhydrin method. The mean sialic acid content found in the ninhydrin technique was 5.58µg/mL, with a more frequent value of 2.70µg/mL. From all 68 HPLC negative samples, two were positive (2.94%) and 66 (97.06%) negative to the colorimetric method. The relative frequency of positive samples was 32%, HPLC had the highest mean serum levels (14.37%), followed by the colorimetric method (5.28%) and acid ninhydrin (3.12%). The high-performance liquid chromatography method was different from the acid and colorimetric ninhydrin methods. The ninhydrin and colorimetric methods were not different from each other, both of which could be used as screening methodologies. Among the three techniques, HPLC was the most sensitive methodology for the detection and quantification of serum in refrigerated raw milk.(AU)

Spectrophotometric Determination of Gentamicin Loaded PLGA Microparticles and Method Validation via Ninhydrin-Gentamicin Complex as a Rapid Quantification Approach.

The purpose of this study was to provide a quantification method with rapid, sensitive, reproducible and cost effective for gentamicin in the form of ninhydrin-gentamicin complex. The utilization of spectrophotometric module to validate the method development for gentamicin loaded microparticles was intended to provide alternative quantification method without undermining the sensitivity of the developed method. The microparticles fabrication process was proven to be suitable in encapsulating gentamicin by using poly(lactic-co-glycolic acid) PLGA without compromising the efficacy of the antibiotic itself. The linearity of 6 known concentrations of ninhydrin-gentamicin complex was obtained with the R2 of 0.9998. The interday and intraday precisions were determined with the acceptance %RSD values of less than 2%. The highest %RSD value was 1.09% which suggested the method to be acceptably precise. The limit of detection (LOD) and limit of quantification (LOQ) values were recorded to be at 0.016 and 0.196 mg/mL respectively. The % recovery of 4 known concentrations indicated the accuracy of the method was high with the recovery range between 98.66% and 101.8%. The parameters analyzed in this study were in accordance with ICH Q2 (R1) guidelines. This quantification method was a promising approach to provide a rapid and cost effective in evaluating gentamicin concentration for in-vitro applications.

Spectrophotometric determination of acyclovir after its reaction with ninhydrin and ascorbic acid.

Acyclovir is a purine-based nucleoside antiviral agent used in the management of Herpes simplex and other viral infections. The present study is aimed at developing and validating a simple and rapid spectrophotometric method for its determination. The mechanism of the proposed method is based on the condensation/coupling reaction between Acyclovir and Ninhydrin-Ascorbic acid at pH 5. A purple colored product with maximum absorption at 540 nm was assayed to quantitatively evaluate the drug content in the formulation. The calibration curve was found to be linear up to 30 μg/ml. Analyte recovery tests carried out by the proposed method gave recovery of between 96.9 – 102.0%. Molar absorptivity and Sandells’ sensitivity were determined to be 41,071.43 L mol-1 cm-1 and 1.84 μg cm-2 respectively. The precision was assessed by determining the inter-day and intra-day variation which ranged between 1.45 – 1.63 % and 0.81 – 1.12 % respectively. The results show that the reaction produced a stable product and the proposed method is cost-effective and possesses adequate accuracy, precision and sensitivity. It can therefore be conveniently applied for the determination of acyclovir in dosage forms.

Spectrophotometric determination of ritonavir by condensation method using ninhydrin and ascorbic acid.

A simple and sensitive extractive visible spectrophotometric method for the assay of Ritonavir in pure and pharmaceutical Formulations based on the reaction between peptide group in RIT and Ninhydrin in the presence of ascorbic acid affords a blue violet coloured product (λ max 560nm). Regression analysis of Beer-Lambert plots showed good correlation in the concentration ranges (20-60) μg/ml. The percent recoveries are obtained as 99.64 ± 0.47 to 100.40 ± 0.45 by proposed method and 99.51 ± 0.25 to 99.92 ± 0.20 by reference method for the formulations respectively. The method can be applied successfully for the estimation of the Ritonavir in the presence of other ingredients that are usually present in formulations. The method offers the advantage of rapidity, simplicity and sensitivity and low cost without the need for expensive instrumentation and reagents.