Spectrofluorimetric determination of cephradine in commercial formulation using ethylacetoacetate-formaldehyde as fluorogenic agent

A simple, precise and sensitive spectrofluorimetric method was developed for the determination of cephradine in pharmaceutical formulations. The method is based on reaction of cephradine with ethyl acetoacetate (EAA) in the presence of formaldehyde in acidic media. The fluorescence intensity of the resultant fluorescent derivative was measured at 443 nm after excitation at 350 nm. The effects of various experimental parameters on the formation of the fluorescent product were studied and optimized. A linear relationship between concentration of cephradine and fluorescence intensity was found over the range of 0.1–5.0 μg/mL. The limit of detection and quantification were calculated an found 1.09 × 10^?2 ± 3.64 × 10^?3 μg/mL and 3.64 × 10^?2 ± 3.64 × 10^?3 μg/mL, respectively and with relative standard deviation (RSD) 4.14%. Accuracy of the method was checked by standard addition method and applied to four different commercial formulations. The percentage recovery was found in the range of 97.03 to 103.80% for Velora suspension, 98.16 to 102.90% for velocef injection, 99.00 to 108.10% for Velora capsule and 99.06 to 104.90% for Cefradin capsule. The good percentage recoveries from all samples indicated that there were no interferences from the common excipients of formulations. The proposed method was successfully applied to the analysis of commercial suspension, capsule and injections. The result obtained by the proposed method was statistically validated with reference HPLC method. No significant difference in the results of the two methods was found regarding accuracy and precision using Student’s t-test and the variance ratio F-test.     

Simultaneous determination of 10 kinds of biogenic amines in rat plasma using high‐performance liquid chromatography coupled with fluorescence detection

We describe a simple, rapid, selective and sensitive HPLC method coupled with fluorescence detection for simultaneous determination of 10 kinds of biogenic amines (BAs: tryptamine, 2‐phenethylamine, putrescine, cadaverine, histamine, 5‐hydroxytryptamine, tyramine, spermidine, dopamine and spermine). BAs and IS were derivated with dansyl chloride. Fluorescence detection (λ_ex/λ_em = 340/510 nm) was used. A satisfactory result for method validation was obtained. The assay was shown to be linear over the ranges 0.005–1.0 μg/mL for tryptamine, 2‐phenethylamine and spermidine, 0.025–1.0 μg/mL for putrescine, 0.001–1.0 μg/mL for cadaverine, 0.25–20 μg/mL for histamine, 0.25–10 μg/mL for 5–hydroxytryptamine and dopamine, and 0.01–1.0 μg/mL for tyramine and spermine. The limits of detection and the limits of quantification were 0.3–75.0 ng/mL and 1.0–250.0 ng/mL, respectively. Relative standard deviations were ≤5.14% for intra‐day and ≤6.58% for inter‐day precision. The recoveries of BAs ranged from 79.11 to 114.26% after spiking standard solutions of BAs into a sample at three levels. Seven kinds of BAs were found in rat plasma, and the mean values of tryptamine, 2‐phenethylamine, putrescine, cadaverine, histamine, spermidine and spermine determined were 52.72 ± 7.34, 11.45 ± 1.56, 162.56 ± 6.26, 312.75 ± 18.11, 1306.50 ± 116.16, 273.89 ± 26.41 and 41.51 ± 2.07 ng/mL, respectively.     

Spectrofluorimetric and Spectrophotometric Determination of Oxamniquine in Pharmaceuticals and Biological Fluids via Derivatization with 4‐Chloro‐7‐Nitrobenzo‐2‐oxa‐1,3‐Diazole (NBD‐Cl)

Spectrophotometric and spectrofluorimetric methods were developed for the determination of oxamniquine (OXM). Both methods are based on coupling with 4‐chloro‐7‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD‐Cl) in borate buffer of pH 7.6, and the reaction product was measured at 400 nm (Method I). The same product was measured by spectrofluorimetry at 480 nm upon excitation at 400 nm (Method II). The absorbance and the fluorescence intensity were enhanced by addition of sodium dodecyl sulphate (SDS). The absorbance‐concentration plot is rectilinear over the range of 5–25 μg/mL with an LOD of 0.31 μg/mL. The fluorescence‐concentration plot is linear over the range of 0.2–1.2 μg/mL with an LOD of 0.03 μg/mL. Both methods were applied to the analysis of capsules, and the results were in good agreement with those obtained using the official method. The method was applied to spiked human plasma; the mean % recovery (n = 5) is 101.05 ± 1.65. A proposal of the reaction pathway is presented.     

Determination of Oxolinic Acid Residues in Gilthead Seabream (Sparus aurata) Muscle Tissue and Plasma by High-Performance Liquid Chromatography

A high-performance liquid chromatographic method for the determination of oxolinic acid (OA) residues in muscle tissue and plasma of the cultured fish gilthead seabream (Sparus aurata L.), is described. OA was extracted with ethyl acetate and after centrifugation the combined extracts were evaporated. To the remaining residue 1 mL of the mobile phase was added and the extract was partitioned with n-pentane which then was rejected by aspiration. OA was chromatographed on a Zorbax^®SB-C₁₈ column at 50^oC and detected by fluorescence detection at λ_ex 327 nm and λ_em 369 nm. The mobile phase was a mixture of 0.1% trifluoroacetic acid (v/v) pH 2.0 and acetonitrile-methanol 3:2 (v/v) in a combination of 50:50 (v/v) and a flow rate of 1.0 mL min^?1, delivered isocratically. Method mean recovery (R%) achieved was 73.7 ± 4.4% (mean ± SD) for blank fortified samples (n=4) range at 50, 100 and 200 μg kg^?1 with a RSD=3.3%. The limit of detection (LOD) was 2.0 μg kg^?1 oxolinic acid in muscle tissue and plasma and the limit of quantification (LOQ) was 5.0 μg kg^?1. The method is fast and suitable to be used with safety and accuracy for the control of OA residues in cultured seabreams and a trained analyst could carry out ready for chromatography more than 50 samples per working day.     

Evaluation and differentiation of the Betulaceae birch bark species and their bioactive triterpene content using analytical FT-vibrational spectroscopy and GC-MS

A new simple, rapid and sensitive reversed-phase liquid chromatographic method was developed and validated for the simultaneous determination of sulpiride (SUL) and mebeverine Hydrochloride (MEB) in the presence of their impurities and degradation products. The separation of these compounds was achieved within 6 min on a 250 mm, 4.6 mm i.d., 5 m particle size Waters^?-C18 column using isocractic mobile phase containing a mixture of acetonitrile and 0.01 M dihydrogenphosphate buffer (45:55) at pH = 4.0. The analysis was performed at a flow rate of 1.0 mL/min with fluorescence-detection at excitation 300 nm and emission at 365 nm. The concentration-response relationship was linear over a concentration range of 10- 100 ng/mL for both MEB and SUL with a limit of detection 0.73 ng/mL and 0.85 ng/mL for MEB and SUL respectively. The proposed method was successfully applied for the analysis of both MEB and SUL in bulk with average recoveries of 100.22 ± 0.757% and 99.96 ± 0.625% respectively, and in commercial tablets with average recoveries of 100.04 ± 0.93% and 100.03 ± 0.376% for MEB and SUL respectively. The proposed method was successfully applied to the determination of MEB metabolite (veratic acid) in real plasma simultaneously with SUL. The mean% recoveries (n = 3) for both MEB metabolite (veratic acid) and SUL were 100.36 ± 2.92 and 99.06 ± 2.11 for spiked human plasma respectively. For real human plasma, the mean% recoveries (n = 3) were and respectively.     

Improved spectrofluorimetric methods for determination of penicillamine in capsules

Two simple, sensitive and specific fluorimetric methods have been developed for the determination of Penicillamine (PNC), a sulphur containing compound. Method (I) involves the reaction of PNC with 2′,7′-bis(acetoxymercuri)-fluorescein (AMF) in the presence of Kolthoff’s buffer, pH 8.2, with subsequent measurement of fluorescence spectra at 520 nm (λ_Ex 497 nm). Method (II) is based on PNC being oxidized into penicillaminic acid using Cerium (IV) in an acidic medium. Method sensitivity has been improved using sodium triphosphate which enhances the luminescence intensity of Ce(III). Fluorescence spectra were then measured at 348 nm (λ_Ex 293 nm). The reaction conditions and the fluorescence spectral properties have been investigated for both methods. Under the described conditions, the proposed methods were applicable over the concentration ranges 0.0048 − 0.0288 μg mL⁻¹ and 0.096 − 0.288 μg mL⁻¹ with mean percentage recoveries 99.95 ± 1.29 and 100.04 ± 1.10 for methods I and II, respectively. The proposed methods were validated in terms of accuracy, precision, LOD and LOQ and robustness and then were successfully applied to the determination of PNC in bulk powder and in capsules as well as in the presence of the related disulphide. The results obtained were determined to be in good agreement with those obtained using a previously reported method.      

Spectrofluorimetric determination of two β-agonist drugs in bulk and pharmaceutical dosage forms via derivatization with dansyl chloride

A sensitive, simple and rapid spectrofluorimetric method has been developed for the determination of both Fenoterol hydrobromide (FNT) and Ritodrine hydrochloride (RTH) in pure and dosage forms. The method is based on derivatization using dansyl chloride (DNS-Cl) as fluorogenic agent and measuring the fluorescence of the products at emission wavelengths of 517 and 515 nm after excitation at 348 and 343 nm for FNT and RTH, respectively. Different experimental parameters affecting the fluorescence intensities were carefully studied and optimized. The relation between fluorescence intensities and drug concentrations were rectilinear over the concentration range of 0.25–6.0 μg/mL for both drugs with a minimum detectability of 0.065 and 0.045 μg/mL for FNT and RTH, respectively. The percentage recoveries ±SD were 100.1 ± 0.9, 99.9 ± 0.6 for FNT and RTH respectively. The proposed method was successfully applied to the determination of both drugs in their commercial dosage forms. The obtained results were statistically validated and agreed well with those obtained with reference methods. A suggestion for the reaction pathway with DNS-Cl was postulated.     

Cationic cyanine as a near-infrared fluorescent probe for the determination of nucleic acids

A new method with a cationic near-IR cyanine as fluorescent probe was developed for the determination of nucleic acids. The near-IR cyanine shows maximum excitation and emission wavelengths at 765 and 790 nm, respectively, in aqueous solution. The method is based on the fluorescence decrease of near-IR cyanine in the presence of nucleic acids. Under optimal conditions, the ratio of fluorescence intensity in the absence and presence of nucleic acids was proportional to the concentration of ¶nucleic acids over the range 0.10–1.2 μg/mL for CT (calf thymus) DNA or SM (salmon sperm) DNA, and 0.10–¶1.6 μg/mL for yeast RNA. The detection limits were ¶30 ng/mL for CT DNA, 25 ng/mL for SM DNA and ¶70 ng/mL for yeast RNA. The relative standard deviation (n = 6) was 2.1% for 500 ng/mL CT DNA, 2.4% for ¶500 ng/mL SM DNA and 2.7% for 500 ng/mL yeast RNA, respectively.     

钙黄绿素荧光法测定痕量钇(Ⅲ)

研究了钙黄绿素荧光法测定Y(Ⅲ)。在pH 8.0的H3BO3-Na2B4O7缓冲溶液和表面活性剂Tween 80的存在下,痕量Y(Ⅲ)能与钙黄绿素反应形成配合物,使体系的荧光强度显著增加,体系的激发和发射波长分别为492和512nm,Y(Ⅲ)的质量浓度在0～2.0μg/mL范围内与△F成良好的线性关系,其线性回归方程为:△F=3.2648+2.384ρ(μg/mL),相关系数r=0.9961,检出限为0.023μg/mL。方法用于乙酸铈中痕量钇(Ⅲ)的测定,测定结果与ICP-AES法相符,加标回收率在96.3%～103.0%之间。     

Kinetic Spectrofluorometric Determination of Thioctic Acid in Bulk and Pharmaceutical Preparations via its Oxidation with Cerium(IV)

A highly simple and sensitive kinetic spectrofluorometric method was developed for the determination of thioctic acid. The method is based on the oxidation of the studied drug with cerium(IV) ammonium sulfate in acidic medium. The fluorescence of the produced Ce(III) was measured at 365 nm after excitation at 255 nm. The different experimental parameters affecting the development and stability of the reaction product were carefully studied and optimized. The method is applicable over the concentration range of 0.02 to 0.12 μg/mL with a detection limit of 6.06 × 10^?3 μg/mL and a quantification limit of 0.02 μg/mL. The method was successfully applied for the assay of the studied drug in pharmaceutical formulations. The results obtained were in good agreement with those obtained with the reference method.     

Quantitative Monitoring of Rutin in Human Urine by Flow Injection‐Chemiluminescence Analysis

In this paper, a rapid and sensitive flow injection‐chemiluminescence (FI‐CL ) method is proposed for the quantitative determination of rutin based on the inhibitory effect of rutin on the chemiluminescence intensity from the luminol–chymotrypsin (CT ) system. The decrease of CL intensity was found to be proportional to the logarithm of rutin concentration in the range 0.1–30.0 ng/mL . A method for the quantification of rutin is proposed, with the limit of detection (LOD ) of 0.03 ng/mL (3σ). A complete analytical process including sampling and washing for rutin determination, which was conducted at a flow rate of 2.0 mL /min, could be performed completely within 30 s, yielding a sample efficiency of 120 h⁻¹. The proposed procedure was successfully applied for the determination of rutin in human urine after oral intake, with recoveries varying from 93.9 to 108.1% and relative standard derivation <4.0% (n = 5). Results showed that urine reached the maximum concentration at ~2.5 h, and the total excretion ratios were (83.5 ± 0.6) and (86.8 ± 0.7)%, respectively, for two volunteers in 8 h. The pharmacokinetic parameters, including the half‐life (1.05 ± 0.02 h), absorption rate constant (1.18 ± 0.01 h⁻¹), and elimination rate constant (0.70 ± 0.01 h⁻¹), were obtained. The possible CL mechanism of the luminol–CT –rutin reaction is discussed by FI‐CL , fluorescence, and molecular docking methods.     

高效液相色谱-荧光分析法同时测定人体尿液中黄蝶呤与异黄蝶呤

建立了高效液相色谱-荧光法同时测定癌症病人尿液中黄蝶呤及异黄蝶呤的新方法。选择荧光检测波长λex=345nm,λem=420nm。以磷酸盐缓冲溶液(pH=7.5)-甲醇(体积比为98∶2)为流动相,流速1.0mL/min,黄蝶呤与异黄蝶呤含量分别在0.0013～0.945μg/mL及0.00017～0.118μg/mL范围内与色谱峰面积呈良好的线性关系,线性相关系数分别为0.9999和0.9996,检出限分别为0.5ng/mL和0.05ng/mL,加标平均回收率在86.2%～107.5%之间。方法应用于癌症病人尿样分析,取得了较好的结果。     

Kinetic and Spectrophotometric Determination of Thioctic Acid in Bulk and Pharmaceutical Formulations

Three simple and sensitive spectrophotometric methods were developed for the determination of thioctic acid in bulk and in its pharmaceutical preparations using iron(III) as an oxidizing agent. Method A is based on kinetic investigation of oxidation reaction of the drug with iron(III) and a subsequent chelation of the produced iron(II) with ferricyanide to form prussian blue colored product at room temperature for a fixed time of 15 minutes at 750 nm. Methods B and C are based on oxidation of the studied drug with iron(III). The equivalent iron(II) produced is allowed to react with either o‐phenanthroline or bipyridyl to give colored species measurable at 510, 522 nm, respectively. Regression analysis of Beer‐Lambert plots showed a good correlation in the concentration ranges of 0.4–4 μg/mL with a detection limit of 0.095 μg/mL for method A and 0.5–5 μg/mL with detection limits 0.137 and 0.127 for method B and C, respectively. The three methods were successfully applied for the determination of the drug in its dosage forms. The percentage recoveries were 99.88 ± 1.40, 99.98 ± 1.26 and 100.64 ± 1.07, respectively.     

A Stability-Indicating Spectrophotometric Method for the Determination of Fenoterol in Pharmaceutical Preparations.

ABSTRACT

A simple and highly sensitive spectrophotometric method was developed for the determination of fenoterol hydrobromide in pharmaceutical formulations. The method is based on measurement of the orange-yellow color peaking at 436 nm, produced when the drug is coupled with diazotized benzocaine in triethylamine medium. The method is applicable over the range of 0.5 – 10 μg/ml, with minimum detectability of 3.6 ng/ml (≈ 9.4×10^?9 M). The molar absorptivity is 4.61×10⁴ L. mol^?1. cm^?1. with relative standard error of 0.196%. The proposed method was successfully applied to the determination of fenoterol in its dosage forms; the mean recoveries of 99.61 ± 1.59 and 98.97 ± 1.52 for tablets and aerosols, respectively, were obtained. The results of the proposed method were favorably compared to those obtained by the official and reference methods. No i8543168 was encountered from the co-formulated drug ipratropium bromide, common excipients and alkaline induced degradation product. A proposal of the reaction pathway is presented.     

New derivatizing reagent for analysis of diethylene glycol by HPLC with fluorescence detection

N‐(2‐Phenyl‐indolyl)‐acetic acid (PIAA), a new fluorescent derivatizing reagent, was used for the determination of diethylene glycol (DEG) by high‐performance liquid chromatography with fluorescence detection. DEG was derivatized to ester by using PIAA in the presence of 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodimide hydrochloride (as dehydrating agent) and 4‐(dimethylamino)pyridine (as base catalyst) in acetonitrile at 60°C for 75 min. The influence of solvent, temperature, catalyst base, concentration of labeling reagent, and couple reagent on the derivatization was investigated. The fluorescence detection was performed with excitation at 340 nm and emission at 377 nm. Baseline separation was obtained on an Ultimate XB‐C18 analytical column with water/acetonitrile gradient elution, good linearity was obtained within 0.5–50 μg/mL with a correlation coefficient of 0.9997. The limit of detection was 0.01 μg/mL (signal‐to‐noise ratio = 3). The method has been successfully applied to determine DEG in toothpaste samples with satisfactory recoveries ranging from 89.0 to 94.9%. The proposed method was shown to be a promising technique for the determination of DEG with high sensitivity.     

Validation of a high-performance chromatographic method for determination of cefotaxime in biological samples

An analytical method for detecting and quantifying cefotaxime in plasma and several tissues is described. The method was developed and validated using plasma and tissues of rats. The samples were analyzed by reversed phase liquid chromatography (HPLC) with UV detection (254 nm). Calibration graphs showed a linear correlation (r > 0.999) over the concentration ranges of 0.5–200 μg/mL and 1.25–25 μg/g for plasma and tissues, respectively. The recovery of cefotaxime from plasma standards prepared at the concentrations of 25 μg/mL and 100 μg/mL was 98.5 ± 3.5% and 101.8 ± 2.2%, respectively. The recovery of cefotaxime from tissue standards of liver, fat and muscle, prepared at the concentration of 10 μg/g was: 89.8 ± 1.2% (liver), 103.9 ± 6.5% (fat) and 97.8 ± 2.1% (muscle). The detection (LOD) and quantitation (LOQ) limits for plasma samples were established at 0.11 μg/mL and 0.49 μg/mL, respectively. The values of these limits for tissues samples were approximately 2.5 times higher: 0.3 μg/g (LOD) and 1.25 μg/g (LOQ). For plasma samples, the deviation of the observed concentration from the nominal concentration was less than 5% and the coefficient of variation for within-day and between-day assays was less than 6% and 12%, respectively. The method was used in a pharmacokinetic study of cefotaxime in the rat and the mean values of the pharmacokinetic parameters are given.     

Application of Boron as the Chromogenic Agent in Spectrophotometry I. A Novel Fading Spectrophotometric Method for the Determination of Aminomethylbenzoic Acid with Boric Acid

It is the first time that boron has been used as a chromogenic agent to determine aminomethylbenzoic acid (PAMBA) by the fading spectrophotometric method in this paper. The study indicates that at pH 10.00 the absorbance of PAMBA decreases when boric acid (BA) is added to the solution. A simple, rapid, sensitive and reliable novel method based on the product of PAMBA and BA is obtained. The stoichiometric ratio of the product is 1:2. Beer's law is obeyed in the range of PAMBA concentrations of 0.13 ? 38.40 μg/mL at a wavelength of 242 nm (ε₂₄₂ is 8.1 × 10⁴ L/mol/cm). The equation of linear regression is A = ‐0.06635 – 0.02914C (× 10^?5 M), with a linear correlation coefficient of 0.9974. The detection limit is 0.12 μg/mL and RSD 0.11%. The method is successfully applied to the determination of PAMBA in pharmaceutical samples, and average recoveries from pharmaceutical samples and urine samples were between 98.4 ? 102.5%. The other components in these samples do not interfere with the determination by the proposed method.     

A comprehensive chromatographic comparison of amphetamine and methylamphetamine extracted from river water using molecular imprinted polymers and without the need for sample derivatization

This work explores the differences between two GCMS instruments for the determination of amphetamine and methylamphetamine extracted from water samples (ultra pure water and river water) without the necessity for derivatization. The instruments contained different generations of gas chromatograph and mass selective detector components and revealed significantly different results when presented with the same samples. The extraction methodology also compared two SPE systems. The extraction efficiency of commercially available molecular imprinted polymers as a sorbent in SPE was compared with commonly used hydrophilic balance sorbent. Molecular imprinted polymers provided excellent recoveries (81 ± 2% and 108 ± 3% at 30 μg L^?1, and 94 ± 2% and 94 ± 2% at 200 μg L^?1 for amphetamine and methylamphetamine, respectively). The best LOD obtained was sufficient for the determination of both drugs extracted from river water (0.029 ± 0.003 and 0.015 ± 0.004 μg L^?1 for amphetamine and methylamphetamine, respectively). These were comparable to literature values obtained through conventional extraction and analysis using LC‐MS/MS but had the advantage of being achieved using an underivatized GCMS method.     

Difference spectrophotemetric assay of mixtures of phenylpropanolamine hydrochloride with guaifensesin or fextromethorphan hydrobromide in olid cough formulations

A difference spectrophotometric method is described for the selective assay of phenylpropanolamine hydrochloride(I) in the presence of guaifenesin(II) or dextromethorphan hydrobromide(III) without prior separation. The method is based on the spectral change upon oxidation of phenylpropanolamine to benzaldehyde by sodium metaperiodate. The difference absorption spectrum is obtained by measuring oxidized against unoxidized phenylpropanolamine. This spectrum exhibits a maximum of 251.5 nm, a minimum at 275 nm and an isosbestic point at 272.5 nm. Absorbance is linear with concentration for 25–100 μg ml^?1 phenylpropanolamine at 251.5 nm. No changes in the spectra of compounds II and III were observed when these compounds were treated with metaperiodate. Guaifenesin and dextromethorphan are assayed by measuring an aliquot of the sample solution against methanol at 281.5 nm and 286 nm, respectively. Phenylpropanolamine does not interfere at these wavelengths. Calibrations are linear over the range 25–125 μg ml^?1 for II and III. Overall recoveries (±SD, n = 5) from simulated tablets were 99.8 ± 2.6% for I and 100.5 ± 0.5% for II; from simulated capsules, the recoveries were 99.2 ± 0.4% for I and 99.6 ± 0.2% for III. The assay was succesfully applied to commercial tablets and capsules containing these compounds.     

Determination of metoprolol in human plasma and urine by high‐performance liquid chromatography with fluorescence detection

A simple, specific and sensitive HPLC method has been developed for the determination of metoprolol in human plasma and urine. Separation of metoprolol and atenolol (internal standard) was achieved on an Ace C₁₈ column (5 μm, 250 mm×4.6 mm id) using fluorescence detection with λ_ex=276 nm and λ_em=296 nm. The mobile phase consists of methanol–water (50:50, v/v) containing 0.1% TFA. The analysis was performed in less than 10 min with a flow rate of 1 mL/min. The assay was linear over the concentration range of 3 – 200 and 5 – 300 ng/mL for plasma and urine, respectively. The LOD were 1.0 and 1.5 ng/mL for plasma and urine, respectively. The LOQ were 3.0 and 5.0 ng/mL for plasma and urine, respectively. The extraction recoveries were found to be 95.6 ± 1.53 and 96.4 ± 1.75% for plasma and urine, respectively. Also, the method was successfully applied to three patients with hypertension who had been given an oral tablet of 100 mg metoprolol.