Fast simultaneous spectrophotometric determination of naphazoline nitrate and methylparaben by sequential injection chromatography

Fast simultaneous determination of naphazoline nitrate and methylparaben in pharmaceuticals using separation method based on a novel reversed-phase sequential injection chromatography (SIC) is described in this contribution as an alternative to classical HPLC. A Chromolith™ Flash RP-18e (25 mm × 4.6 mm) column (Merck^®, Germany) and a FIAlab^® 3000 system (USA) with a six-port selection valve and 5.0 ml syringe pump were used for sequential injection chromatographic separations in our study. The mobile phase used was methanol/water (40:65, v/v), pH 5.2 adjusted with triethylamine 0.8 μl ml⁻¹ and acetic acid, at flow rate 0.9 ml min⁻¹. UV detection provided by DAD detector and two wavelengths were simultaneously monitored for increasing sensitivity of determination. Detector was set up at 220 nm for naphazoline nitrate and 256 nm for methylparaben and ethylparaben (IS). There is no necessity to use pre-adjustment of sample of nasal drops (only dilution with mobile phase) so the time of the whole analysis is very short. The validation parameters have shown good results: linearity of determination for both components (naphazoline nitrate and methylparaben), correlation coefficient >0.999; repeatability of determination (R.S.D.) in the range 0.5-1.6% at three different concentration levels, detection limits 0.02 μg ml⁻¹ (naphazoline nitrate) and 0.20 μg ml⁻¹ (methylparaben and ethylparaben), and recovery from the pharmaceutical preparations in the range 100.06-102.55%. The chromatographic resolution between peaks of compounds was more than 4.0 and analysis time was less than 4 min under the optimal conditions. The advantages and drawbacks of SIC against classical HPLC are discussed showing that SIC can be an advantageous alternative in many cases.     

A novel application of Onyx™ monolithic column for simultaneous determination of salicylic acid and triamcinolone acetonide by sequential injection chromatography

A novel and fast simultaneous determination of triamcinolone acetonide (TCA) and salicylic acid (SA) in topical pharmaceutical formulations by sequential injection chromatography (SIC) as an alternative to classical high performance liquid chromatography (HPLC) has been developed. A recently introduced Onyx™ monolithic C18 (50 mm × 4.6 mm, Phenomenex^®) with 5 mm monolithic precolumn were used for the first time for creating sequential injection chromatography system based on a FIAlab^® 3000 with a six-port selection valve and 5.0 mL syringe pump in study. The mobile phase used was acetonitrile/water (35:65, v/v), pH 3.3 adjusted with acetic acid at flow rate 0.9 mL min⁻¹. UV detection provided by fibre-optic DAD detector was set up at 240 nm. Propylparaben was chosen as suitable internal standard (IS). There is only simple pre-adjustment of the sample of topical solution (dilution with mobile phase) so the analysis is not uselessly elongated. Parameters of the method showed good linearity in wide range, correlation coefficient >0.999; system precision (relative standard deviation, R.S.D.) in the range 0.45-1.95% at three different concentration levels, detection limits (3σ) 1.00 μg mL⁻¹ (salicylic acid), 0.66 μg mL⁻¹ (triamcinolone acetonide) and 0.33 μg mL⁻¹ (propylparaben) and recovery from the pharmaceutical preparations in the range 97.50-98.94%. The chromatographic resolution between peaks of compounds was more than 4.5 and analysis time was 5.1 min under the optimal conditions. The advantages of sequential injection chromatography against classical HPLC are discussed and showing that SIC can be a method of option in many cases.     

Non-equilibrium determination of metoclopramide and tetracaine hydrochloride by sequential injection spectrophotometry

A new sequential injection spectrophotometric method was proposed for the determination of metoclopramide and tetracaine hydrochloride. The method was based on the detection of an unstable red intermediate compound resulting from the reaction of metoclopramide or tetracaine hydrochloride with potassium dichromate, in the presence of sodium oxalate, in sulfuric acid solution. The related reaction mechanisms of this new method have been studied. The experimental conditions were optimized for the stopped-flow and continuous-flow sequential injection models. For continuous flow, the linear range for determination of metoclopramide, the detection limit and the sampling frequency were 13-130 μg ml⁻¹, 9.4 μg ml⁻¹ and 40 samples per hour, respectively. For stopped flow, they were 3-42 μg ml⁻¹, 1.0 μg ml⁻¹ and 18 h⁻¹, respectively. Adopting the continuous-flow model for tetracaine hydrochloride, the linear range was 25-300 μg ml⁻¹, and the detection limit was 18.0 μg ml⁻¹ with sampling frequency of 40 h⁻¹. This method has been used to determine metoclopramide and tetracaine hydrochloride in pharmaceutical preparations, and the results are compared with those determined by the pharmacopoeia method. Statistical analysis reveals that there was no evidence of significant difference between the methods.     

Determination of pesticides fenoxycarb and permethrin by sequential injection chromatography using miniaturized monolithic column

Sequential injection chromatography system equipped with miniaturized 10 mm monolithic column was used for fast simultaneous determination of two pesticides—fenoxycarb (FC) and permethrin (PM). The system was composed of a commercial sequential injection analysis (SIA) system (FIAlab^® 3000, 6-port selection valve and 5.0 mL syringe pump), commercially available column Chromolith™ RP-18e (10 mm × 4.6 mm i.d.) (Merck^®, Germany) and CCD UV-vis detector (USB 2000, Ocean-optics) with 1.0 cm Z-flow cell, absorbance was monitored at 225 nm. The mobile phase used for analysis was acetonitrile/water (60:40, v/v), flow rates were 0.6 mL min⁻¹ for elution of fenoxycarb and 1.2 mL min⁻¹ for elution of permethrin. For each analysis 4.8 mL of mobile phase was used. The chromatographic resolution between both compounds was >8 and analysis time was <6.5 min under the optimal conditions. Limits of detection were determined at 2.0 μg mL⁻¹ for fenoxycarb and 1.0 μg mL⁻¹ for permethrin. Samples were prepared by diluting with mobile phase and injected volume was 10 μL for each analysis. Developed method was applied to analysis of both pesticides in veterinary pharmaceutical foams and sprays ARPALIT^® Neo (Aveflor, Czech Republic). SIC method was compared with validated method (HPLC, reverse phase 100 mm monolithic column, gradient elution).     

Evaluation of a novel PTFE material for use as a means for separation and preconcentration of trace levels of metal ions in sequential injection (SI) and sequential injection lab-on-valve (SI-LOV) systems: Determination of cadmium(II) with detection by electrothermal atomic absorption spectrometry (ETAAS)

The operational characteristics of a novel poly(tetrafluoroethylene) (PTFE) bead material, granular Algoflon^®, used for separation and preconcentration of metal ions via adsorption of on-line generated non-charged metal complexes, were evaluated in a sequential injection (SI) system furnished with an external packed column and in a sequential injection lab-on-valve (SI-LOV) system. Employed for the determination of cadmium(II), complexed with diethyldithiophosphate (DDPA), and detection by electrothermal atomic absorption spectrometry (ETAAS), its performance was compared to that of a previously used material, Aldrich PTFE, which had demonstrated that PTFE was the most promising for solid-state pretreatments. By comparing the two materials, the Algoflon^® beads exhibited much higher sensitivity (1.6107 μg l⁻¹ versus 0.2956 μg l⁻¹ per integrated absorbance (s)), and better retention efficiency (82% versus 74%) and enrichment factor (20.8 versus 17.2), although a slightly smaller linear dynamic range (0.05-0.25 μg l⁻¹ versus 0.05-1.00 μg l⁻¹). Moreover, no flow resistance was encountered under the experimental conditions used. The results obtained on three standard reference materials were in good agreement with the certified values.     

Development and validation of a HPLC method for the determination of buprenorphine hydrochloride, naloxone hydrochloride and noroxymorphone in a tablet formulation

A simple isocratic reversed-phase high-performance liquid chromatographic method (RP-HPLC) was developed for the simultaneous determination of buprenorphine hydrochloride, naloxone hydrochloride dihydrate and its major impurity, noroxymorphone, in pharmaceutical tablets. The chromatographic separation was achieved with 10 mmol L⁻¹ potassium phosphate buffer adjusted to pH 6.0 with orthophosphoric acid and acetonitrile (17:83, v/v) as mobile phase, a C-18 column, Perfectsil Target ODS3 (150 mm × 4.6 mm i.d., 5 μm) kept at 35 °C and UV detection at 210 nm. The compounds were eluted isocratically at a flow rate of 1.0 mL min⁻¹. The average retention times for naloxone, noroxymorphone and buprenorphine were 2.4, 3.8 and 8.1 min, respectively. The method was validated according to the ICH guidelines. The validation characteristics included accuracy, precision, linearity, range, specificity, limit of quantitation and robustness. The calibration curves were linear (r > 0.996) over the concentration range 0.22-220 μg mL⁻¹ for buprenorphine hydrochloride and 0.1-100 μg mL⁻¹ for naloxone hydrochloride dihydrate and noroxymorphone. The recoveries for all three compounds were above 96%. No spectral or chromatographic interferences from the tablet excipients were found. This method is rapid and simple, does not require any sample preparation and is suitable for routine quality control analyses.     

The development of sequential injection analysis coupled with lab-on-valve for copper determination

A sequential injection analysis (SIA) using lab-on-valve with air segmentation and spectrophotometric detection was designed for copper(II) determination. It is based on the reaction of copper(II) and 2-carboxy-2′-hydroxy-5′-sulfoformazyl benzene (Zincon) in a weak alkaline solution between the air zones. Beer's Law was obeyed over the range of 0.1-2.0 mg L⁻¹ copper(II) with a correlation coefficient 0.9985 and a slope of 0.2893 absorbance unit/mg L⁻¹. The relative standard deviation was 2.0% for a series of 10 measurements of 0.5 mg L⁻¹ copper(II) solution. The detection limit (3 S/N) and the limit of quantification (LOQ) were 0.05 and 0.17 mg L⁻¹ respectively. This method has been successfully applied to determination of copper(II) in wastewater with a sample throughput of 120 h⁻¹. The method is superior to the batchwise method in that it provides fully automation, rapidity, less reagents and sample consumption with little waste generation.     

Two-column Sequential Injection Chromatography—New approach for fast and effective analysis and its comparison with gradient elution chromatography

This work presents novel approach in low-pressure chromatography flow systems—two-column Sequential Injection Chromatography (2-C SIC) and its comparison with gradient elution chromatography on the same instrument. The system was equipped with two different chromatographic columns (connected to selection valve in parallel design) for isocratic separation and determination of all components in composed anti-inflammatory pharmaceutical preparation (tablets). The sample was first injected on the first column of length 30 mm where less retained analytes were separated and then the sample was injected on the second column of length 10 mm where more retained analytes were separated. The SIC system was based on a commercial SIChrom™ manifold (8-port high-pressure selection valve and medium-pressure syringe pump with 4 mL reservoir) (FIAlab^®, USA) with two commercially available monolithic columns the “first column” Chromolith^® Flash RP-18e (25 mm × 4.6 mm i.d. with guard column 5 mm × 4.6 mm i.d.) and the “second column” Chromolith^® RP-18e (10 mm × 4.6 mm i.d.) and CCD UV-vis detector USB 4000 with micro-volume 1.0 cm Z flow cell. Two mobile phases were used for analysis (one for each column). The mobile phase 1 used for elution of paracetamol, caffeine and salicylic acid (internal standard) was acetonitrile/water (10:90, v/v, the water part of pH 3.5 adjusted with acetic acid), flow rate was 0.9 mL min⁻¹ (volume 3.0 mL of mobile phase per analysis). The mobile phase 2 used for elution of propyphenazone was acetonitrile/water (30:70, v/v); flow rate was 1.2 mL min⁻¹ (volume 1.5 mL of mobile phase per analysis). Absorbance was monitored at 210 nm. Samples were prepared by dissolving of one tablet in 30% acetonitrile and 10 μL of filtered supernatant was injected on each column (2 × 10 μL). The chromatographic resolution between all compounds was >1.45 and analysis time was 5.5 min under the optimal conditions. Limits of detection were determined at 0.4 μg mL⁻¹ for paracetamol, at 0.5 μg mL⁻¹ for caffeine and at 0.7 μg mL⁻¹ for propyphenazone. The new two-column chromatographic set-up developed as an alternative approach to gradient elution chromatography shows evident advantages (time and solvent reduction more than one-third) as compared with single-column gradient SIC method with Chromolith^® Flash RP-18 (25 mm × 4.6 mm i.d. with guard column 5 mm × 4.6 mm i.d.).     

Spectrophotometric determination of magnesium in pharmaceutical preparations by cost-effective sequential injection analysis

A simple and rapid, inexpensive spectrophotometric method was proposed for magnesium assay in pharmaceutical preparations by sequential injection analysis (SIA). The method is based on the reaction between o-cresolphthalein complexone (CPC) and Mg(II) in alkaline media, yielding a pink colored complex with absorption maximum at 570 nm. Since the formation constant between Ca-CPC and Mg-CPC is similar, initially a sample/standard solution was aspirated into the holding coil followed by a mixture of masking-buffer solutions. This was done because masking of calcium should be accomplished before Mg-CPC complexation. Then the reagent was introduced into the reaction coil to produce a colored complex, which is measured spectrophotometrically at 570 nm. In this way the interference of calcium was reduced. Furthermore, all the parameters that affect the reaction were evaluated. The calibration curve is linear over a range of 0-20 mg l⁻¹ of Mg(II) with a detection limit of 0.24 mg l⁻¹. A sample throughput of 80 samples per hour and relative standard deviation <2.0% were achieved. The proposed method was successfully applied for the assay of magnesium in three different compositions of pharmaceutical preparations (tablets). The results were found to be in good agreement with the manual flame atomic absorption spectrophotometry (FAAS) and UV-Vis spectrophotometry methods and with the claimed values by the manufactures. The t-test shows no significant difference at 95% confidence level.     

Sequential injection method for the direct spectrophotometric determination of bismuth in pharmaceutical products

A spectrophotometric method is reported for the determination of bismuth in pharmaceutical products using sequential injection analysis. Methylthymol blue (MTB) was used as a color forming reagent and the absorbance of the Bi(III)-MTB complex was monitored at 548 nm. The various chemical and physical variables that affected the reaction were studied. A linear calibration graph was obtained in the range 0.0-75.0 mg l⁻¹ Bi(III) at a sampling frequency of 72 h⁻¹. The reagent consumption was considerably reduced compared to conventional flow injection systems, as only 150 μl of MTB were consumed per run. The precision was very satisfactory (s_r=0.5%, at 50.0 mg l⁻¹ Bi(III), n=12) and the limit of detection, c_L, was 0.250 mg l⁻¹. The developed method was applied successfully to the analysis of various pharmaceutical products containing Bi(III). The relative errors e_r, were <1.5% in all cases and were evaluated by comparison of the obtained results with those found using atomic absorption spectrometry as the reference method.     

Sequential injection spectrophotometric determination of phenylephrine hydrochloride in pharmaceutical preparations

A fully automated sequential injection spectrophotometric method for the determination of phenylephrine hydrochloride in pharmaceutical preparations is reported. The method is based on the condensation reaction of the analyte with 4-aminoantipyrine in the presence of potassium ferricyanide. The absorbance of the condensation product was monitored at 503 nm. A linear relationship between the relative peak height and concentration was obtained in the range 0.5-17.5 mg l⁻¹. The detection limit (as 3σ value) was 0.09 mg l⁻¹ and repeatability was 0.8 and 0.6% at 2.5 and 5 mg l⁻¹, respectively. Results obtained by this method agreed very well with those obtained by the AOAC official method.     

Simple automated generation of gradient elution conditions in sequential injection chromatography using monolithic column

The paper deals with the concept of simple automated creation of gradient profile of the mobile phase for gradient-elution sequential injection chromatography (GE-SIC). The feasibility and merits of this concept are demonstrated on the separation and simultaneous assay of indomethacin as active principle and of its two degradation products (5-methoxy-2-methylindoleacetic acid and 4-chloro-benzoic acid) in a topical pharmaceutical formulation.The GE-SIC separation was performed with a FIAlab^® 3000 SIC set-up (USA) equipped with an Onyx™ Monolithic C18 (25 mm × 4.6 mm, Phenomenex^®) column, a six-port selection valve, a 5-mL syringe pump and a fiber-optics UV CCD detector. Ketoprofen was used as an internal standard (IS). The gradient elution was achieved by automated reproducible mixing of acetonitrile and aqueous 0.2% phosphoric acid in the holding coil of the SIC system. Different profiles of the gradient elution were tested. The optimal gradient using two mobile phases 30:70 and 50:50 of acetonitrile/0.2% phosphoric acid (v/v) was achieved under the optimum flow rate 1.2 mL min⁻¹. The chromatographic resolution R between the peaks of all solutes (including the IS) was >2.00. The repeatability of retention times was characterized by the RSD values 0.18-0.30% (n = 6). Net separation time was 3.5 min and the mobile phase consumption was 4.5 mL for a single GE-SIC assay. The figures of merit of the novel GE-SIC method compared well with those of conventional HPLC.     

Normal spectrophotometric and stopped-flow spectrofluorimetric sequential injection methods for the determination of alendronic acid, an anti-osteoporosis amino-bisphosphonate drug, in pharmaceuticals

A normal spectrophotometric and a stopped-flow (SF) spectrofluorimetric method have been developed and optimized for the determination of alendronic acid (ALD) in its pharmaceutical formulations. Both methods are automated using the sequential injection analysis (SIA) principle. The spectrophotometric assay is based on the reaction of the analyte with Cu(II) ions in acidic medium to form an UV-absorbing derivative (λ_max = 240 nm). The SF spectrofluorimetric method is based on the reaction of ALD with o-phthalaldehyde (OPA) in the presence of 2-mercaptoethanol at basic medium (λ_ex = 340 nm/λ_em = 455 nm). Linear calibration curves were obtained in the range 1.0-60.0 mg l⁻¹ ALD for the UV method, and in the range 0.13-10.0 mg l⁻¹ ALD for the SF spectrofluorimetric one. The sampling rates were 60 and 30 h⁻¹, respectively. The developed assays are critically compared and their advantages are discussed. Both methods were applied to the analysis of an ALD containing pharmaceutical formulation with satisfactory accuracy and precision.     

A reagent-free method based on a photo-induced fluorimetry in a sequential injection system

According to the current demands of environmentally friendly analytical chemistry and with a view to achieving lower reagent consumption with improved analytical performance, an automatic methodology composed of a photoreactor and fluorimetric detection (λ_exc = 287 nm, λ_em = 378 nm) was developed. To this end, a sequential injection analysis (SIA) system was developed for indomethacin determination using ultra-violet (UV) light which promotes an increase in the fluorescence of indomethacin. This increase in sensitivity makes it possible to apply this methodology to a dissolution test and to determine indomethacin in pharmaceutical formulations.The calibration graph for indomethacin was linear between 4.10 × 10⁻⁶ and 9.00 × 10⁻⁵ mol L⁻¹and the detection limit was 1.23 × 10⁻⁶ mol L⁻¹. The method was proven to be reproducible with a R.S.D. < 5% and sampling rate of approximately 20 per hour. The potential effect of several compounds commonly used as excipients on analytical signals was studied and no interfering effect was observed. Statistical evaluation at the 95% confidence level showed good agreement between the results obtained for the pharmaceutical samples with both the SIA system and comparison batch procedures.     

Automation of simultaneous release tests of two substances by sequential injection chromatography coupled with Franz cell

This presented paper deals with a methodology for the separation and simultaneous determination of two active substances in topical pharmaceutical formulation composed of lidocaine (L) and prilocaine (P). The methodology described is based on the sequential injection chromatography (SIC) with UV detection. Monolithic Column Chromolith Flash RP-18, 25 mm × 4.6 mm (Merck, Germany) was used. Separation was performed using elution with binary mobile phase composed of acetonitrile-phosphate buffer 0.05 M (40:80 (v/v)) + 0.01% triethylamine (adjusted to pH 7.1 with H₃PO₄) at a flow rate of 0.6 ml min⁻¹. The analysis duration was <7 min. The method was linear over the range of 2.5-200 mg l⁻¹ with a detection limit of 0.25 mg l⁻¹ for both substances.The system was then coupled with Franz cell. Fully automated system for the in vitro release testing of semisolid dosage forms based on sequential injection analysis (SIA) was developed. Simultaneous measurement of L and P release was done by this system. Samples were taken in 10.5 min intervals during 4 h of the release test. Each test was followed by calibration with five standard solutions. Receiving medium was replenished automatically by the system.     

11-Molybdobismuthophosphate—A new reagent for the determination of ascorbic acid in batch and sequential injection systems

The guanidinium salt of the new heteropolymolybdate 11-molybdobismuthophosphate Gua₆PBiMo₁₁O₄₀ (11-MBP) was synthesized, characterized and used as a reagent for batch spectrophotometric (SP) and sequential injection determination of ascorbic acid (AsA). When compared to other Keggin's heteropolyanions, the reduction of 11-MBP with AsA is both fast and maximal within a pH range of 1.6-2.0. The stoichiometry of the reaction was determined using molar ratio and continuous variation methods and was shown to be 1:1. The molar absorptivity of the reduced form of 11-MBP was 6.0 × 10³ L mol⁻¹ cm⁻¹ at 720 nm. The reaction is also specific for AsA. Only cysteine, hydroquinone and hydroxyacids were found to interfere with the reaction, while no interference was observed with the common reducing agents, including reducing sugars, catecholamines, nitrite, sulfite and iron(II) ions. Batch SP and sequential injection analysis (SIA) systems were developed for the determination of AsA, with calibration ranges of the SP methods at 2 × 10⁻⁶-8 × 10⁻⁵ M for a 10 mm cell and 5 × 10⁻⁷-3 × 10⁻⁵ M for a 50 mm cell and a limit of detection at 3 × 10⁻⁷ M. The linear range of the SIA method was 6 × 10⁻⁶-5 × 10⁻⁴ M, with a detection limit of 2 × 10⁻⁶ M and a sample throughput of 15 h⁻¹. The proposed methods were successfully used for the determination of AsA in both pharmaceuticals and fruit juices, and the results were consistent with those provided by the 2,6-dichlorophenolindophenol method.     

Implementing stepwise solvent elution in sequential injection chromatography for fluorimetric determination of intracellular free amino acids in the microalgae Tetraselmis gracilis

The concept of sequential injection chromatography (SIC) was exploited to automate the fluorimetric determination of amino acids after pre-column derivatization with o-phthaldialdehyde (OPA) in presence of 2-mercaptoethanol (2MCE) using a reverse phase monolithic C₁₈ stationary phase. The method is low-priced and based on five steps of isocratic elutions. The first step employs the mixture methanol: tetrahydrofuran: 10 mmol L⁻¹ phosphate buffer (pH 7.2) at the volumetric ratio of 8:1:91; the other steps use methanol: 10 mmol L⁻¹ phosphate buffer (pH 7.2) at volumetric ratios of 20:80, 35:65, 50:50 and 65:35. At a flow rate of 10 μL s⁻¹ a 25 mm long-column was able to separate aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), serine (Ser), glutamine (Gln), glycine (Gly), threonine (Thr), citruline (Ctr), arginine (Arg), alanine (Ala), tyrosine (Tyr), phenylalanine (Phe), ornithine (Orn) and lysine (Lys) with resolution >1.2 as well as methionine (Met) and valine (Val) with resolution of 0.6. Under these conditions isoleucine (Ile) and leucine (Leu) co-eluted. The entire cycle of amino acids derivatization, chromatographic separation and column conditioning at the end of separation lasted 25 min. At a flow rate of 40 μL s⁻¹ such time was reduced to 10 min at the cost of resolution worsening for the pairs Ctr/Arg and Orn/Lys. The detection limits varied from 0.092 μmol L⁻¹ for Tyr to 0.51 μmol L⁻¹ for Orn. The method was successfully applied to the determination of intracellular free amino acids in the green alga Tetraselmis gracilis during a period of seven days of cultivation. Samples spiked with known amounts of amino acids resulted in recoveries between 94 and 112%.     

An emphatic orthogonal signal correction-support vector machine method for the classification of tissue sections of endometrial carcinoma by near infrared spectroscopy

A sequential injection analysis (SIA) spectrophotometric method for determining tetracycline (TC), chlortetracycline (CTC) and oxytetracycline (OTC) in different sample matrices were described. The method was based on the reaction between tetracyclines and yttrium (III) in weak basic micellar medium, yielding the light yellow complexes, which were monitored at 390, 392 and 395 nm, respectively. A cationic surfactant, cetyltrimethylammonium bromide (CTAB) was used to obtain the micellar system. The linear ranges of calibration graphs were between 1.0 × 10⁻⁵ and 4 × 10⁻⁴ mol L⁻¹, respectively. The molar absorptivities were 5.24 × 10⁵, 4.98 × 10⁴ and 4.78 × 10⁴ L mol⁻¹ cm⁻¹. The detection limits (3σ) were between 4.9 × 10⁻⁶ and 7.8 × 10⁻⁶ mol L⁻¹ whereas the limit of quantitations (10σ) were between 1.63 × 10⁻⁵ and 2.60 × 10⁻⁵ mol L⁻¹ the interday and intraday precisions within a weak revealed as the relative standard deviations (R.S.D., n = 11) were less than 4%. The method was rapid with a sampling rate of over 60 samples h⁻¹ for the three drugs. The proposed method has been satisfactorily applied for the determination of tetracycline and its derivatives in pharmaceutical preparations together with their residues in milk and honey samples collected in Chiang Mai Province. The accuracy was found to be high as the Student's t-values were found to be less than the theoretical ones. The results were compared favorably with those obtained by the conventional spectrophotometric method.     

Flow injection fluorescence determination of dopamine using a photo induced electron transfer (PET) boronic acid derivative

An automated flow injection analysis system was developed for the fluorometric determination of dopamine in pharmaceutical injections. The method is based on the quenching effect of dopamine on m-dansylaminophenyl boronic acid (DAPB) fluorescence due to the reverse photo induced electron transfer (PET) mechanism. Effects of pH and interfering species on the determination of dopamine were examined. Calibration for dopamine, based on quenching data, was linear in the concentration range of 1.0 × 10⁻⁵ to 1.0 × 10⁻⁴ M. Detection limit (3 s) of the method was found to be 3.7 × 10⁻⁶ M. Relative standard deviation of 1.2% (n = 10) was obtained with 1.0 × 10⁻⁵ M dopamine standard solution. The proposed method was applied successfully for the determination of dopamine in pharmaceutical injection sample. The sampling rate was determined as 24 samples per hour.     

Automated measurement of permeation and dissolution of propranolol HCl tablets using sequential injection analysis

Aim of this study was to automate sampling and quantification of the previously described apparatus for combined determination of dissolution and permeation through Caco-2 monolayer by means of sequential injection analysis (SIA). Native fluorescence of propranolol HCl in Krebs-Ringer buffer (KRB) was used for quantification. Sampling was done at three different locations within the apparatus at a high sampling frequency (approximately 60 h⁻¹). Injection volume delivered to the fluorescence detector was 50 μL for permeation monitoring and 25 μL for dissolution monitoring. Linear regression for 50 μL injection yielded a detection limit calculated as 0.04 μg mL⁻¹ of propranolol HCl in KRB (R² > 0.999). However, linearity for dissolution monitoring was not given for the complete range of concentrations and first order polynomial calibration was established (R² > 0.9999). To conclude, the SIA system was able to monitor simultaneously dissolution and permeation of the immediate release propranolol HCl tablets and the authors succeeded in automating the apparatus for combined measurement of dissolution and permeation. In addition, the obtained data was consistent with data obtained by manual sampling followed by HPLC analysis.