Automated derivatization and fluorimetric determination of biogenic amines in milk by zone fluidics coupled to liquid chromatography.

A novel zone-fluidics/high pressure liquid chromatographic (ZF-HPLC) method is described for the simultaneous determination of six biogenic monoamines, in the presence of hexylamine as internal standard. Automated on-line derivatization of the analytes with naphthalene-2,3-dicarboxaldehyde/cyanide ions was performed using the ZF concept and the derivatives were injected on the HPLC column for separation/detection. The influence of the ZF operation conditions on the derivatization reaction was investigated. The isoindoles formed were separated on a Kromasil C18 column (250 mm × 4 mm i.d., 5 µm), using an isocratic mobile phase of methanol/water (80:20, v/v) at a flow rate of 1.0 mL min(-1). Monitoring and quantification was carried out by fluorescence detection at 424/494 nm. The limits of detection were at the pg level with a sample volume of 20 µL. The whole procedure was evaluated and fully validated for the determination of biogenic amines in milk samples.

Zwitterionic hydrophilic interaction chromatography coupled with post-column derivatization for the analysis of glutathione in wine samples.

In this study the development, validation and application of a new chromatographic method for the determination of glutathione (GSH) in wine samples is presented. The separation of the GSH was carried out using a sulfobetaine-based hydrophilic interaction chromatography (HILIC) analytical column whereas its detection was carried out spectrofluorimetrically (λext/λem=340/455 nm) after post-column derivatization with o-phthalaldehyde. GSH was separated efficiently from matrix endogenous compounds of wines by using a mobile phase of 15 mmol L(-1) CH3COONH4 (pH=2.5)/CH3CN, 35/65% (v/v). The parameters of the post-column reaction (pH, amount concentration of the reagent and buffer solution, flow rate, length of the reaction coil) were investigated. The linear determination range for GSH was 0.25-5.0 µmol L(-1) and the LOD was 19 nmol L(-1). No matrix effect was observed, while the accuracy was evaluated with recovery experiments and was ranged between 89% and 108%.

Selective fluorimetric method for the determination of histamine in seafood samples based on the concept of zone fluidics.

In the present article we report our results on the development of a selective automated method for the determination of histamine in seafood using the concept of zone fluidics. The method is based on the sequential on-line reaction of the analyte with o-phthalaldehyde in the absence of a nucleophilic reagent, followed by acidification. The careful selection of the chemical and instrumental variables enabled the determination of the analyte with adequate sensitivity at the low micromolar level and with specificity against other biogenic amines and amino acids such as histidine. The LOD was 0.05 µmol L(-1) (0.6 mg kg(-1)) and linearity was obeyed in the range of 0.5-15 µmol L(-1) (5.5-170 mg kg(-1)). The proposed method offers a satisfactory sampling rate of 15 h(-1) and adequate accuracy and precision for the analysis of seafood products after minimum sample preparation and without employing a separation technique.

Determination of glutathione and cysteine in yeasts by hydrophilic interaction liquid chromatography followed by on-line postcolumn derivatization.

In the present study, we report a new method for the determination of two primary thiols, cysteine (CYS) and glutathione (GSH), by hydrophilic interaction LC. The polar analytes are separated isocratically using a mobile phase consisting of 65% acetonitrile/35% ammonium acetate (15 mmol/L, pH 2.0) and are detected at 285 nm following on-line postcolumn derivatization by the thiol-selective reagent methyl propiolate. The main figures of merit included linearity in the range of 5-200 µmol/L and an LOD 0.6 µmol/L for both compounds. The absence of matrix effect allowed the determination of CYS and GSH in various yeast samples. GSH was present in most of the samples at levels ranging between 0.9 and 3.1 mg/g, whereas CYS was determined in only one sample at a significantly lower concentration. In terms of accuracy, the percent recoveries ranged between 91.2 and 105.6% for GSH, and 91.6 and 106.9% for CYS.

On-line cleavage of disulfide bonds by soluble and immobilized tris-(2-carboxyethyl)phosphine using sequential injection analysis.

Reduction of disulfide bonds is - in many cases - a critical pretreatment step for the determination of thiols in real samples. This study reports the first systematic investigation of the potentials of the on-line reduction of disulfide bonds under flow conditions in a sequential injection setup. One of the most promising reducing agents, tris-(2-carboxyethyl)phosphine (TCEP) was selected for this purpose while the Ellman's disulfide (DTNB) was used as model compound. The study involved the investigation of several parameters that affected the kinetics and efficiency of the reaction, including stopped-flow experiments. Both soluble and immobilized TCEP on agarose beads were examined. The results confirmed that both forms of TCEP can be used as an advantageous on-line reducing reagent for disulfide bonds under flow conditions.

Automated determination of total captopril in urine by liquid chromatography with post-column derivatization coupled to on-line solid phase extraction in a sequential injection manifold.

The present study reports a new liquid chromatographic (HPLC) method for the determination of the anti-hypertension drug captopril (CAP) in human urine. After its separation from the sample matrix in a reversed phase HPLC column, CAP reacts with the thiol-selective reagent ethyl-propiolate (EP) in a post-column configuration and the formed thioacrylate derivative is detected at 285 nm. Automated 4-fold preconcentration of the analyte prior to analysis was achieved by an on-line solid phase extraction (SPE) step using a sequential injection (SI) manifold. The Oasis HLB SPE cartridges offered quantitative recoveries and effective sample cleaning by applying a simple SPE protocol. The limits of detection and quantitation were 10 µg L(-1) and 35 µg L(-1) respectively. The percent recoveries for the analysis of human urine samples ranged between 90 and 96% and 95 and 104% using aqueous and matrix matched calibration curves respectively.

Automated pre-column derivatization of thiolic fruit-antibrowning agents by sequential injection coupled to high-performance liquid chromatography using a monolithic stationary phase and an in-loop stopped-flow approach.

The present study reports the very first application of ethyl propiolate (EP) as an advantageous pre-column derivatization reagent for the determination of thiols by liquid chromatography (LC). Cysteine (CYS), glutathione (GSH) and N-acetylcysteine (NAC) were derivatized online under stopped-flow conditions in a sequential injection (SI) system coupled to HPLC. The formed derivatives were separated isocratically with a monolithic stationary phase (100×4.6 mm id) and UV detected at 285 nm. Critical parameters that affected the online pre-column derivatization reaction (e.g. the reaction time and the amount concentration of EP) and the separation (e.g. pH and the composition of the mobile phase) were investigated. The developed analytical scheme offers a total analysis time of less than 10 min, limits of detection in the range of 0.24-0.35 µmol/L and satisfactory linearity up to 200 µmol/L for all analytes. The proposed method was applied to the analysis of the selected thiols--that are often employed as antibrowning agents--in fresh fruit samples.

Rapid determination of methylxanthines in real samples by high-performance liquid chromatography using the new FastGradient narrow-bore monolithic column.

The present study reports one of the very first analytical applications of the new narrow-bore monolithic column, FastGradient Chromolith (50mm x 2.0mm i.d.). The three major methylxanthines (theobromine, theophylline and caffeine) were separated rapidly and determined simultaneously in various real samples. Based on the unique characteristics of this novel monolithic column the analytes were separated efficiently (R(s)>3.0) in less than 5min at a low flow rate of 0.5mLmin(-1) and using a low volume fraction of organic solvent (5% acetonitrile (ACN) in water) in the mobile phase. UV detection was carried out at 274nm. The separation was optimized in terms of mobile phase composition, flow rate and injection volume, while the method was validated for linearity, detection and quantitation limits, within and day-to-day precision, accuracy and ruggedness. Its applicability was demonstrated by analyzing a variety of real samples including beverages, soft drinks, herbal products and pharmaceuticals. Compared to a well-established monolithic (Performance Chromolith 100mm x 4.6mm i.d.) and a particulate reversed phase column (Hypersil ODS 5microm 150mm x 4.6mm i.d.), the narrow-bore FastGradient column offered satisfactory performance, faster analysis time and drastic reduction in the consumption of mobile phase and organic solvents.

Automated zone-sampling dilution by coupling sequential injection analysis to high-throughput HPLC for the direct determination of gemfibrozil.

A novel automated analytical scheme for the rapid determination of gemfibrozil in drug dissolution samples is reported. The procedure is based on direct coupling of a low pressure continuous flow technique such as sequential injection analysis (SI) to HPLC. SI performs automated dilution of the samples based on zone sampling and fills on-line the loop of the high pressure injection valve. Rapid separation of the analyte from the samples' matrix can be achieved in less than 1.0 min, by using a short RP monolithic column (25x4.6 mm id) at a flow rate of 2 mL/min. The SI and HPLC parts of the setup operate independently: during HPLC separation the next sample is treated by SI. This way a maximum throughput of 60 samples per hour is achieved allowing the complete analysis of a batch of six dissolution samples within 12-18 min based on the replicates. The proposed method was validated in terms of linearity, LOD and LOQ, precision, selectivity and accuracy. Its applicability was tested during quality control of four validation batches of a gemfibrozil-containing formulation. The results were in good agreement with the HPLC method proposed by the US Pharmacopeia.

Ethyl-propiolate as a novel and promising analytical reagent for the derivatization of thiols: study of the reaction under flow conditions.

The present investigation demonstrates the potentials of ethyl-propiolate (EP), a novel derivatizing reagent for thiols. To the best of our knowledge this is the first systematic study of EP in analytical chemistry. The reaction was investigated under flow conditions using sequential injection (SI) analysis and UV detection at 285 nm. The reaction kinetics was affected by parameters such as the pH, the concentration of EP and the temperature and was thoroughly examined exploiting stopped-flow experiments. Cysteine (CYS) and captopril (CAP) were selected as model thiolic compounds in terms of chemical structures. Finally, the applicability of EP as a derivatization reagent for analytical purposes was demonstrated by the development and validation of a novel automated assay for the determination of CAP in pharmaceuticals.

Automated sample preparation coupled to sequential injection chromatography: on-line filtration and dilution protocols prior to separation.

Sequential injection chromatography (SIC) is a valuable tool in analytical chemistry as it can combine the automation capabilities of low pressure continuous flow techniques and the separation power of HPLC into a single instrumental configuration. The present study reports an automated SI setup allowing on-line filtration and dilution of the samples before separation through a short monolithic column. The applicability of the procedure was evaluated by studying the behavior of acyclovir formulations under forced degradation conditions. Minimal sample preparation is required prior to analysis. Thorough validation of the on-line dilution SIC assay was carried out and proved its validity in terms of critical parameters such as precision, accuracy and robustness. The results were evaluated by parallel experiments and analysis using the procedure recommended by the USP based on conventional HPLC using particulate-based column.

Hybrid sequential injection-flow injection manifold for the spectrophotometric determination of total sulfite in wines using o-phthalaldehyde and gas-diffusion.

A new automated spectrophotometric method for the determination of total sulfite in white and red wines is reported. The assay is based on the reaction of o-phthalaldehyde (OPA) and ammonium chloride with the analyte in basic medium under SI conditions. Upon on-line alkalization with NaOH, a blue product is formed having an absorption maximum at 630 nm. The parameters affecting the reaction - temperature, pH, ionic strength, amount concentration and volume of OPA, amount concentration of ammonium chloride, flow rate and reaction coil length - and the gas-diffusion process - sample and HCl volumes, length of mixing coil, donor flow rate - were studied. The proposed method was validated in terms of linearity (1-40 mgL(-1), r=0.9997), limit of detection (c(L)=0.3 mgL(-1)) and quantitation (c(Q)=1.0 mgL(-1)), precision (s(r)=2.2% at 20 mgL(-1) sulfite, n=12) and selectivity. The applicability of the analytical procedure was evaluated by analyzing white and red wine samples, while the accuracy as expressed by recovery experiments ranged between 96% and 106%.

Separation and determination of nimesulide related substances for quality control purposes by micellar electrokinetic chromatography.

A micellar electrokinetic chromatography (MEKC) method has been developed and validated for the determination of nimesulide related compounds in pharmaceutical formulations. Electrophoretic separation of six European Pharmacopoeia (EP) impurities (A-F) was performed using a fused silica capillary (L(eff.)=50 cm, L(tot.)=57 cm, 50 microm i.d.) with a background electrolyte (BGE) containing 25 mM borate buffer (pH 9.5), 30 mM sodium dodecyl sulphate and phi=3% (v/v) acetonitrile. The influence of several factors (surfactant and buffer concentration, pH, organic modifier, applied voltage, capillary temperature and injection time) was studied. The method was suitably validated with respect to linearity, limit of detection and quantification, accuracy, precision and selectivity. The calibration curves obtained for the six compounds were linear over the range 5-12 microgml(-1) (0.05-0.12%). The relative standard deviations (s(r)) of intra- and inter-day experiments were less than 5.0%. The detection limits ranged between 0.7 and 1.6 microgml(-1) depending on the impurity. The proposed method was applied successfully to the quantification of nimesulide impurities in its pharmaceutical formulation.

Selective determination of cyanides by gas diffusion-stopped flow-sequential injection analysis and an on-line standard addition approach.

A highly selective sequential injection (SI) method for the automated determination of weak-acid-dissociable cyanides is reported. The analytical procedure is based on the on-line reaction of the analyte with ninhydrin in carbonate medium to form a coloured product (lambda(max)=510 nm). Cyanides are removed from sample matrix by acidification through a gas-diffusion step incorporated in the SI manifold. The effect of instrumental and chemical variables was studied. By adopting an on-line standard addition protocol, the sensitivity of the proposed method was enhanced drastically, without affecting the determination range. The assay was validated in terms of linearity (up to 200 microg L(-1)), limit of detection (c(L)=2.5 microg L(-1)), limit of quantitation (c(Q)=7.5 microg L(-1)), precision (s(r)<2.5% at 100 microg L(-1)) and selectivity. High tolerance against critical species such as sulfides and thiocyanates was achieved. The applicability of the method was demonstrated by analyzing tap and mineral water samples at levels below the limits established by international E.U. and U.S. organizations. The percent recoveries were satisfactory in all cases, ranging between 94.2 and 103.6%.

Optimization and validation of a dissolution test for selegiline hydrochloride tablets by a novel rapid HPLC assay using a monolithic stationary phase.

The present study reports the optimization and validation of a dissolution test for selegiline.HCl tablets using a new high-performance liquid chromatographic (HPLC) method. Rapid separation of the analyte from sample matrix was achieved in less than 60s using a Cromolith RP-18e monolithic column using UV detection at 220 nm. Thorough validation of the assay based on pre-defined criteria included linearity, LOD/LOQ, accuracy, precision, selectivity and ruggedness. The dissolution test was optimized in terms of dissolution medium, basket (type I)/paddle (type II) agitation and rotation speed. Its ruggedness was also validated. The presented analytical and dissolution procedures are currently being applied in the quality and stability control of Cosmopril tablets (5mg/tablet selegiline.HCl, Cosmopharm Ltd., Korinthos, Greece).

Development and validation of a high-throughput high-performance liquid chromatographic assay for the determination of caffeine in food samples using a monolithic column.

The present study reports the development and validation of a high-throughput high-performance liquid chromatographic (HPLC) assay for the determination of caffeine in food samples. The analyte was separated rapidly from sample matrix using a short monolithic column (50 mm x 4.6 mm i.d.). The flow rate was 3.0 mL min(-1), while the mobile phase consisted of ACN/water (10:90, v/v). Caffeine was detected directly at 274n m. Under the optimal HPLC conditions, the sampling rate was 60 h(-1). The assay was validated for linearity, LOD and LOQ, precision, selectivity and ruggedness. The case of external calibration versus standard addition for the analysis of real samples was also examined. The proposed assay was applied to the analysis of beverages and coffee samples.

Review of recent applications of flow injection spectrophotometry to pharmaceutical analysis.

Pharmaceutical analysis is one of the most important fields in analytical chemistry. The discovery of new drugs and the on-going update of international regulations for the safety and efficacy of pharmaceutical formulations demand the continuous development of new analytical methods. Inevitably, automation plays an important role, especially when a lot of samples have to be analyzed in the minimum of time. The present study reviews the applications of flow injection (FI) spectrophotometry to the determination of active pharmaceutical ingredients (APIs) in their respective formulations. However, the topic covered in this study is important not only to pharmaceutical analytical scientists. The principles, figures of merit and "chemistry" of the presented methods can be of interest to bio-analytical and clinical chemists as well for the analysis of biological samples, to environmental analysts that study the up-to-date demand of the determination of the fate of pharmaceuticals in the environment and even to toxicologists and forensic scientists. This review covers scientific contributions published later than 2000. A variety of FI procedures based on homogeneous (direct UV measurements, colour-forming reactions, metal-drug interactions) and heterogeneous (optical sensors and solid-phase reactors) systems are discussed. A third section covers on-line sample pretreatment (solid-phase extraction, liquid-liquid extraction, on-line digestion, etc.).

Automated determination of flutamide by a validated flow-injection method: application to dissolution studies of pharmaceutical tablets.

The first flow-injection (FI) method for the determination of flutamide--a potent antiandrogen used for the treatment of prostate cancer--is reported. The method is based on the direct measurement of the absorbance of the analyte at 310 nm under flow conditions. Parameters affecting the determination such as detection wavelength, sample injection volume and flow rate were studied and optimized. The assay was validated (linearity, limits of detection and quantitation, accuracy, repeatability, reproducibility and selectivity) for the dissolution studies of flutamide-containing tablets during stability testing. The results were in good agreement with high performance liquid chromatography (HPLC) used as a reference method.

Validated high-throughput HPLC assay for nimesulide using a short monolithic column.

High samples analysis rate is a key demand in modern pharmaceutical analysis, especially during new product development and validation of industrial-scale manufacturing process. The present study reports a validated HPLC assay for the dissolution studies of nimesulide-containing tablets (Lizepat 100 mg/tab, Cosmopharm Ltd., Korinthos, Greece). Using a 50 mm x 4.6 mm i.d. monolithic column (Chromolith, Merck) and acetonitrile-phosphate buffer (pH 7.0; 10 mM) (34:66, v/v) as the mobile phase, the separation cycle was completed in 60s at a flow rate of 4.0 ml min(-1). The assay was validated in terms of selectivity against potential impurities of the active ingredient, detection and quantification limits, linearity, accuracy and inter-/intra-day precision. Results from the application of the HPLC method to the accelerated and long-term dissolution stability control of Lizepat tablets (Lot 005) are reported.

High-throughput HPLC assay of acyclovir and its major impurity guanine using a monolithic column and a flow gradient approach.

Acyclovir and its major impurity guanine are determined rapidly by the incorporation of a monolithic column (100 mm x 4.6 mm i.d., Merck) to an automated HPLC system. A simple flow gradient protocol was adopted in order to accelerate the separation-detection cycle. Using 0.2% CH(3)COOH (pH 3.1) as the mobile phase and detection at 254 nm, guanine was effectively separated from the system peak (t(R)=1.25 min), while the retention time of acyclovir was 2.35 min. Linearity of the assay was validated in the range 0.1-1.0% guanine and 80-120% acyclovir (n=5). The accuracy and within- and day-to-day precision of the method were also validated, while the limits of detection and quantitation of both analytes were determined. The proposed method was successfully applied to the quality control of acyclovir raw material and the quality and stability control of acyclovir-containing pharmaceutical creams (Hagevir 5%, w/w, Cosmopharm Ltd., Korinthos, Greece).