Identification, trace level quantification, and in silico assessment of potential genotoxic impurity in Famotidine.

Potential genotoxic impurities in medications are an increasing concern in the pharmaceutical industry and regulatory bodies because of the risk of human carcinogenesis. To prevent the emergence of these impurities, it is crucial to carefully examine not only the final product but also the intermediates and key starting material (KSM) used in drug synthesis. During the related substances analysis of KSM of Famotidine, an unknown impurity in the range of 0.5-1.0% was found prompting the need for isolation and characterization due to the possibility of its to infiltrate into the final product. In this study, the impurity was isolated and characterized as 5-(2-chloroethyl)-3,3-dimethyl-3,4-dihydro-2H-1,2,4,6-thiatriazine 1,1-dioxide using multiple instrumental analysis, uncovering a structural alert that raises concern. Considering the potential impact of impurity on human health, an in silico genotoxicity assessment was established using Derek and Sarah tool in accordance with ICH M7 guideline. Furthermore, molecular docking and molecular dynamics simulation were performed to evaluate the specific interaction of the impurity with DNA. The findings reveal consistent interaction of the impurity with the dG-rich region of the DNA duplex and binding at the minor groove. Both in silico prediction and molecular dynamic study confirmed the genotoxic character of the impurity. The newly discovered impurity in famotidine has not been reported previously, and there is currently no analytical method available for its identification and control. A highly sensitive HPLC-UV method was developed and validated in accordance with ICH requirements, enabling quantification of the impurity at trace level in famotidine ensuring its safe release.

An eco-friendly multi-analyte high-performance thin layer chromatographic densitometric determination of amoxicillin, metronidazole, and famotidine in their ternary mixtures and simulated gastric juice: A promising protocol for eradicating Helicobacter pylori.

Gastrointestinal tract disorders constitute a heavy burden to healthcare providers. To eradicate Helicobacter pylori infection, different triple therapy protocols have been proposed. Among which are combinations of proton pump inhibitors (e.g., omeprazole), histamine-2 receptor antagonists (e.g., famotidine), along with antibiotics (e.g., amoxicillin). In this work, a sensitive and accurate high-performance thin-layer chromatographic method was developed for the simultaneous determination of amoxicillin, metronidazole, and famotidine in bulk powder and laboratory-prepared combined-tablet mixtures. Complete separation of the cited compounds was achieved using pre-coated silica gel plates with a mixture of methanol:chloroform:toluene:water:glacial acetic acid (5:2:1.5:0.5:0.1 v/v/v/v/v) as the mobile phase. The method was fully validated as per the international conference of harmonization guidelines. Good linearity, a correlation coefficient of 0.9991, was obtained in the concentration ranges 0.1-1.6 µg/band (amoxicillin), 0.1-0.9 µg/band (metronidazole), and 0.1-0.9 µg/band (famotidine). Since the method allowed the determination of the three compounds in combined tablets with a high degree of selectivity, accuracy, precision, with cost-effectiveness, it could be used for regular quality control. Moreover, the applicability of the proposed method was extended to the determination of the ternary mixture in simulated gastric juice. Method greenness was assessed using different green metrics.

Simultaneous interaction, degradation, and kinetic study of sparfloxacin with H2 receptor antagonist.

Sparfloxacin is a quinolone carboxylic acid derivative that shows activity as an antimicrobial agent, against a wide range of Gram-negative and Gram-positive organisms. It is clinically useful for the treatment of urinary tract infections, respiratory tract infections and gynecological infections. In this study in vitro drug-drug interaction of sparfloxacin has been carried out with famotidine and ranitidine. For these studies a two-component spectrophotometric process has been developed for sparfloxacin assay in the presence of famotidine or ranitidine. The reproducibility of the method is within ±5%. The technique has been applied to the development of sparfloxacin in methanol. The interaction studies of sparfloxacin with ranitidine and famotidine were carried out in methanol and methanol: Water mixtures (30:70, v/v; 50:50, v/v) and the kinetics of sparfloxacin degradation were evaluated in the presence and absence of famotidine and ranitidine. The decrease in the rate of degradation of sparfloxacin in the presence of famotidine or ranitidine, compared to that of sparfloxacin alone, indicated the possibility of interaction between the sparfloxacin and famotidine or ranitidine. The Thin layer chromatography (TLC) of the degraded solution showed the presence of a degradation product of sparfloxacin. The studies show that complexation with famotidine or ranitidine may affect the bioavailability of sparfloxacin.

Development and validation of RP-HPLC method for simultaneous determination of cefpodoxime proxetil and H2 receptor antagonists in pharmaceutical dosage forms.

A new method on RP-HPLC is devised and validated, as per ICH guidelines, for the synchronous estimation of cefpodoxime proxetil and H2-receptor antagonits that are Cimetidine, Famotidine and Ranitidine. The method is simple, accurate, expeditious, reproducible, robust and precise. Chromatography was done on a C18 (250 x 4.6mm) column with methanol: water as mobile phae in the ratio of 70:30 (v/v), pumped at a flow rate of 1ml/min and pH was maintained using 85% ortho-phosphoric acid at 3. The λ max 240 nm was preferred for UV detection. A good linear relationship was attained, over the concentration ranges of 20-70 µg/ml and 5-30µg/ml, for cefpodoxime proxetil and H2 blockers respectively, with a correlation coefficient of R= 0.9987 to 0.9992. The method was validated and found precised (i.e. intra day and interday analysis) with RSD <2%. LOD and LOQ observations were under 0.4806 to 2.6069µg/ml which proved the method to be sensitive. The method provided satisfactory results of robustness and reproducibility, when validated and applied successfully for analysis of dosage forms.

Derivative emission spectrofluorimetry: Application to the analysis of newly approved FDA combination of ibuprofen and famotidine in tablets.

A new combination of ibuprofen (NSAID) and famotidine (H2 receptor antagonist) was recently approved by the FDA. It was formulated to relief pain while decreasing the risk of ulceration, which is a common problem for patients receiving NSAID. A rapid and simple derivative emission spectrofluorimetric method is proposed for the simultaneous analysis of this combination in their pharmaceutical preparation. The method is based upon measurement of the native fluorescence intensity of the two drugs at λex = 233 nm in acetonitrile. The emission data were differentiated using the first (D1) derivative technique. The plots of derivative fluorescence intensity versus concentration were rectilinear over a range of 2-35 and 0.4-8 µg/mL for both ibuprofen (IBU) and famotidine (FAM), respectively. The method was sensitive as the limits of detection were 0.51 and 0.12 µg/mL and limits of quantitation were 1.70 and 0.39 µg/mL, for IBU and FAM respectively. The proposed derivative emission spectrofluorimetric method was successfully applied for the determination of the two drugs in their synthetic mixtures and tablets with good accuracy and precision. The proposed method was validated as per ICH guidelines.

Development and validation of chromatographic methods for simultaneous determination of ibuprofen and famotidine in presence of related substances in pharmaceutical formulations.

Two validated methods for the simultaneous determination of ibuprofen and famotidine in the presence of ibuprofen impurity (4-isobutylacetophenone) and or famotidine degradation products were described. The first method was a simple TLC method where separation was performed on silica gel platesusing ethyl acetate: methanol: ammonia (9:2:1, by volume) as a mobile phase. Rf values were found to be 0.40, 0.94, 0.66, 0.27, 0.83 for ibuprofen, 4-isobutylacetophenone, famotidine, famotidine acid and basic degradation products, respectively. The second method is by HPLC on C18 column using methanol: phosphate buffer pH 3 (80:20, v/v) as a mobile phase. Retention times were found to be 2.2, 9.9, and 8.6 for famotidine, ibuprofen, and 4-isobutylacetophenone, respectively. Both methods were validated according to the ICH guidelines and applied for the determination of the two drugs in pure powder and combined dosage form without interference from the excipients.

Stability-Indicating HPLC Method for Determination of Ibuprofen and Famotidine Degradation Products.

A new stability-indicating high-performance liquid chromatographic method for the quantitative determination of ibuprofen and famotidine degradation products in combined pharmaceutical products was developed and validated. The current aim of this study is to develop a rapid, accurate and robust analytical stability indicating impurity method that can separate ibuprofen, famotidine and their related impurities by using a reversed-phase high-performance liquid chromatography. A Zorbax SB-Phenyl column (4.6 × 150 mm2, 5-µm particle size) with mobile phase containing phosphate buffer solution with a pH value of 3.0 and acetonitrile was used. The flow rate was 0.8 mL/min and the analytes were detected by UV detector at 265 nm. The retention times of ibuprofen and famotidine were 18.43 and 5.14 min, respectively. This method was validated to confirm specificity, linearity, sensitivity (limit of detection and limit of quantitation), precision, accuracy, robustness and sample stability according to the International Conference on Harmonization guidelines. Studies have been completed and reported with two active substances in the combined dosage form and seven impurities in total. There is no method in the literature that simultaneously distinguishes and quantitatively analyzes both active substances and degradation products.

[Stability of Six Drugs in Total Parenteral Nutrition Solution].

Elneopa NF No. 1 and No. 2 infusions are total parenteral nutrition solutions packaged in four-chambered infusion bags. They have been used as home parenteral nutrition, with various drugs injected into the infusion bags, for treating patient symptoms. In this study, we investigated the stability of six drugs, including famotidine, scopolamine butylbromide, furosemide, bromhexine hydrochloride, betamethasone sodium phosphate, and metoclopramide hydrochloride in the infusion bags under dark conditions at 4℃ for 7 days. Additionally, we developed a high-performance liquid chromatography method to determine drug concentrations in the infusions. The concentrations of injected famotidine, scopolamine butylbromide, and betamethasone sodium phosphate remained unchanged when the four chambers of Elneopa NF No. 1 and No. 2 were opened and the infusions were mixed. Their respective concentrations in the upper and lower chambers also remained unchanged. The concentration of furosemide in the upper chamber of the No. 1 infusion bag decreased after 5 days, although no change was observed in the other chambers and the mixed infusions with the four chambers opened. The concentration of bromhexine hydrochloride slightly decreased in the upper chambers (approximately 3%) after the co-infusion but decreased significantly in the other chambers and the mixed infusions with the four chambers opened. The concentration of metoclopramide hydrochloride significantly decreased in the upper chambers after the co-infusion; however, no change in concentration was observed in the other chambers and the mixed infusion with the four chambers opened. The results of this study provide useful information on home-based parenteral nutrition.

Validated HPTLC method for simultaneous quantitation of paracetamol, diclofenac potassium, and famotidine in tablet formulation.

A sensitive, simple, selective, precise, and accurate HPTLC method of analysis for paracetamol, diclofenac potassium, and famotidine both as a bulk drug and in tablet formulation was developed and validated. The method used HPTLC aluminum plates precoated with silica gel 60F254 as the stationary phase, and the mobile phase consisted of toluene-acetone-methanol-formic acid (5 + 2 + 2 + 0.01, v/v/v/v). Densitometric evaluation of the separated zones was performed at 274 nm. This system was found to give compact spots for paracetamol (Rf value = 0.62 +/- 0.03), diclofenac potassium (0.75 +/- 0.02), and famotidine (0.17 +/- 0.03). The linear regression analysis data for the calibration plots showed a good linear relationship over the concentration range of 1625-9750 ng/spot for paracetamol, 250-1500 ng/spot for diclofenac potassium, and 100-600 ng/spot for famotidine. The method was validated for precision, robustness, and recovery according to International Conference on Harmonization guidelines. No chromatographic interference from the tablet excipients was found. Statistical analysis showed that the method was repeatable and selective for the simultaneous quantitation of the three drugs in tablet formulation and for routine quality control of raw materials of the drugs.

Pentabromobenzyl-RP versus triazole-HILIC columns for separation of the polar basic analytes famotidine and famotidone: LC method development combined with in silico tools to follow the potential consequences of famotidine gastric instability.

The competence of hydrophilic interaction (HILIC) and reversed phase liquid chromatography (RPLC) modes, employing two new stationary phases: triazole- and pentabromobenzyl-bonded silica (PBr), respectively, was inspected for separation of two polar basic analytes: famotidine (FAM) and its acidic degradant famotidone (FON). Comparison of the chromatographic efficiency, greenness, and economy aspects showed that the RPLC is superior to the HILIC. Hence, the RPLC method was adopted and validated adhering to the FDA guidelines showing excellent linearity for FAM (1.0-20.0 µg/mL) with a detection limit of 0.14 µg/mL. The method was applied to study the behavior of FAM in simulated gastric juice (SGJ), where it exhibited rapid degradation yielding FON. This degradation pathway is a probable major reason for the poor bioavailability of FAM. The kinetic study of the gastric degradation of FAM in SGJ demonstrated pseudo-first order reaction with a rate constant of 8.1 × 10-3 min-1. Moreover, FAM degradation has been proven to be pH-dependent and catalyzed by the gastric juice components. Hence, in situ buffered dosage form is recommended to overcome or decrease this problem. Molecular docking study shows that FON is missing a crucial stabilizing interaction with the key amino acid Asp98 causing a reduced activity at hH2R receptor relative to FAM. Moreover, ADMET properties prediction revealed some differences in the toxicity, pharmacokinetics, metabolism, and solubility profiles of FAM and FON.

Spectrofluorimetric determination of famotidine in pharmaceutical preparations and biological fluids. Application to stability studies.

A simple, economic, selective, and stability indicating spectrofluorimetric method was developed for the determination of famotidine (FMT); is based on its reaction with 9, 10-phenanthraquinone in alkaline medium to give a highly fluorescent derivative measured at 560 nm after excitation at 283 nm. The fluorescence intensity-concentration plot was rectilinear over the concentration range of 50-600 ng/ml with minimum quantification limit (LOQ) of 13.0 ng/ml and minimum detection limit (LOD) of 4.3 ng/ml. The factors affecting the development of the fluorescence intensity of the reaction product were carefully studied and optimized. The method was applied for the determination of FMT in its dosage forms. The stability of the compound was studied, and the proposed method was found to be stability indicating one. The results obtained were in good agreement with those obtained by the official method. Furthermore, the method was applied for the determination of FMT in spiked and real human plasma. The mean % recovery (n = 4) was found to be 99.94 +/- 0.24, and 105.13 +/- 0.64 for spiked and real human plasma, respectively. The composition of the reaction product as well as its stability constant was also investigated. Moreover, the method was utilized to investigate the kinetics of both alkaline and oxidative induced degradation of the drug. The apparent first order rate constant and half life time of the degradation product was calculated. A proposal of the reaction pathway was postulated.

Quantitative determination of famotidine polymorphs: X-ray powder diffractometric and Raman spectrometric study.

X-ray powder diffractometric and Raman spectrometric methods were developed for quantitative measurement of the polymorphic forms of famotidine in their mixtures. This study aims to deduce some useful conclusions regarding quantitative polymorph analysis, which could also be utilized in industrial practice. Both form A and form B of famotidine possess specific X-ray diffraction reflections as well as characteristic Raman vibrational bands, which permits simple determination of the phases in their mixtures. Keeping in mind that multivariate data processing by chemometric approach is thought of nowadays as superior over univariate one, the results of the two evaluation methods were compared by precision, accuracy as well as robustness. It was found that both approaches provide similar results provided analytically useful data regions are properly selected. Overcoming the common problems of quantitative X-ray powder diffractometry and solid state Raman spectrometry both permit accurate quantification of famotidine polymorphs; the latter, however, seems to be more favourable in regular laboratory practice.

Spectrophotometric determination of H(2)-receptor antagonists via their oxidation with cerium(IV).

A simple, accurate and sensitive spectrophotometric method has been developed and validated for determination of H(2)-receptor antagonists: cimetidine, famotidine, nizatidine and ranitidine hydrochloride. The method was based on the oxidation of these drugs with cerium(IV) in presence of perchloric acid and subsequent measurement of the excess Ce(IV) by its reaction with p-dimethylaminobenzaldehyde to give a red colored product (lambda(max) at 464nm). The decrease in the absorption intensity of the colored product (DeltaA), due to the presence of the drug was correlated with its concentration in the sample solution. Different variables affecting the reaction were carefully studied and optimized. Under the optimum conditions, linear relationships with good correlation coefficients (0.9990-0.9994) were found between DeltaA values and the concentrations of the drugs in a concentration range of 1-20microgml(-1). The assay limits of detection and quantitation were 0.18-0.60 and 0.54-1.53microgml(-1), respectively. The method was validated, in terms of accuracy, precision, ruggedness and robustness; the results were satisfactory. The proposed method was successfully applied to the determination of the investigated drugs in pure and pharmaceutical dosage forms (recovery was 98.3-102.6+/-0.57-1.90%) without interference from the common excipients. The results obtained by the proposed method were comparable with those obtained by the official methods.

Multivariate Validated Models for Simultaneous Determination of Ibuprofen and Famotidine in the Presence of Related Substances.

Two multivariate validated spectrophotometric methods, namely partial least-squares (PLS) and principal component regression (PCR), were developed and validated for the determination of ibuprofen and famotidine in presence of famotidine degradation products and ibuprofen impurity (4-isobutylacetophenone). A calibration set was prepared in which the two drugs together with the degradation products and impurity were modeled using a multilevel multifactor design. This calibration set was used to build the PLS and PCR models. The proposed models successfully predicted the concentrations of both drugs in validation samples, with low root mean square error of cross validation (RMSECV) percentage. The method was validated by the estimate of the figures of merit depending on the net analyte signal. The results of the two models showed that the simultaneous determination of both drugs could be performed in the concentration ranges of 100-500 µg/mL for ibuprofen and 5-25 µg/mL for famotidine. The proposed multivariate calibration methods were applied for the determination of ibuprofen and famotidine in their pharmaceutical formulation, and the results were verified by the standard addition technique.

Application of polyacrylonitrile nanofibers decorated with magnetic carbon dots as a resonance light scattering sensor to determine famotidine.

In this study, a novel resonance light scattering (RLS) sensor was synthesized using polyacrylonitrile nanofibers decorated with magnetic carbon dots (MCDs@NFs) nanocomposite and applied for famotidine (FMD) determination. The MCDs@NFs nanocomposite was synthesized by combining electrospinning and a simple one-step hydrothermal method. Different methods were applied in order to characterize the MCDs@NFs nanocomposite such as: scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FT-IR), Transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD). Light scattering properties of the synthesized nanocomposite in the presence or absence of FMD have been selected as the detection signal considering the fact that FMD addition increases the RLS intensities of the system. Thus, the prepared nanocomposite was employed as a RLS sensor to detect FMD. A linear response was observed under the optimal conditions in range of 0.15-50.0µmolL-1 with detection limit of 0.04µmolL-1. The MCDs@NFs nanocomposite was effectively capable in determining FMD in real samples and the results were close to those results obtained by reversed-phase HPLC method (RP-HPLC).

Development of a Validated Comparative Stability-Indicating Assay Method for Some H2-Receptor Antagonists.

A comparative force degradation high performance thin layer chromatography (HPTLC) method was developed and validated for some H2-receptor antagonists. The studied H2-receptor antagonists were ranitidine (RAN), nizatidine (NIZ) and famotidine (FAM). The degradation behaviors of the studied H2-receptor antagonists were studied under different stress conditions (hydrolytic, thermal and oxidative) conditions as well as storage conditions according to International Conference on Harmonization (ICH) recommendations. A stability-indicating HPTLC method was optimized in order to separate the analyte from the degradation products formed under various stress conditions. Full separation of the drugs from their degradation products was successfully achieved on an HPTLC precoated silica gel plates. Densitometric measurements were carried out using a Camag TLC Scanner III in the absorbance mode at 320 nm for RAN and NIZ, and 280 nm for FAM. The limits of detection and limits of quantitation range were 5.47-9.37 and 16.30-31.26 ng/band, respectively, for all investigated drugs. The validation studies were performed according to ICH requirements. The developed method was simple, rapid and reliable hence it could be applied for routine quality control analysis of the investigated H2-receptor antagonists in dosage forms. The kinetic behavior, degradation rate constants and half-lives of the degradation of the investigated drugs were studied and compared at different stress conditions. The present study provides, for the first time, a new vision to compare the degradation kinetics of H2-receptor antagonists at the same degradation procedures.

Sensitive determination of G-protein-coupled receptor binding ligands by solid phase extraction-electrospray ionization-mass spectrometry.

High affinity Histamine H2-receptor binding ligands were assayed by automated solid phase extraction (SPE) coupled via electrospray ionization with a Quadrupole-Time-of-Flight mass spectrometer (Q-ToF-MS). The mass spectrometric behavior of these analytes was tested in aqueous solutions with several (nine) volatile salts, in different pH, and with various methanol contents. Out of the high amount of available ligands, three fluorescent-labeled molecules (5706, 5707, and 5708) were studied in detail. The limits of detection (LODs) for all three compounds obtained in mass spectrometric detection was 1 fmol (absolute) in continuous flow and FIA (flow injection analysis) measurements. The results obtained with FIA-fluorescence detection gave LODs a factor 10-100 times higher. A systematic investigation of sample solving conditions, loading flow conditions, and elution flow conditions made the automated SPE-MS coupling efficient. Ideally, the ligands were dissolved in MeOH-25 mM phosphate buffer (30:70 v/v; pH 11), the SPE loading flow comprised MeOH-25 mM phosphate buffer (30:70 v/v; pH 11) and the SPE elution flow contained MeOH-100 mM ammonium formate solution (90:10 v/v; pH 3). Using this method on a C18-modified silica cartridge (C18, 5 microm, 100 A, 300 microm i.d. x 5 mm, LC Packings) assures high recovery and achieved LODs for all three compounds of 5 fmol (absolute). As an absolute amount of ligands specifically bound on H2-receptors in biochemical experiments is, as will be published elsewhere, between 10 and 100 fmol, the SPE-MS method for the basic compounds can be directly applied for these Histamine H2-receptors.

Rapid and reliable determination of illegal adulterant in herbal medicines and dietary supplements by LC/MS/MS.

In recent years, dietary supplements and herbal medicines are increasing in popularity all over the world. However, it is problematic that some manufacturers illegally included synthetic drugs in their products. Due to the extremely complex matrices of those products, most existing methods for screening illegal adulterations are time-consuming and liable to false positive. In this paper, a robust LC/MS/MS method for the high-throughput, sensitive and reliable determination of illegal adulterations from herbal medicines and dietary supplements was established. Minimal LC separation was employed and MRM was used to simultaneously monitor the three transitions under their respective optimal collision energy for each compound. Positive results were determined only if well-defined peaks appeared at all of the three transitions and the ratios among the peak areas were within given threshold. In this study, the method had been applied for the screening of nine most commonly adulterated therapeutic substances, such as sildenafil (Viagra) and famotidine, and the lower limits of detection of these compounds ranged from 0.05 to 1.5 ng/ml. Little sample preparation was needed for this method and the analysis time was less than 5 min/sample. The reliability has been demonstrated by the test with blank matrix. Over 200 products that were under suspicion by SDA of China had been assayed and till now no false negative or positive result was found. This method is rapid, simple, reliable and capable of screening multiple adulterants in one run.

Stability of metronidazole, tetracycline HCl and famotidine alone and in combination.

Metronidazole, tetracycline HCl and famotidine are commonly used for the treatment of Helicobacter pylori-associated peptic ulcer. In this paper, stabilities of these drugs and their combinations in solid and liquid states were studied as part of preformulation in the development of a combination drug delivery system. Solubility studies of metronidazole and tetracycline HCl were investigated, which indicated that both metronidazole and tetracycline HCl have high solubilities at and around pH 2.0. Metronidazole is relatively stable with little degradation in liquid phase. Tetracycline HCl in the dry state is stable when stored at room temperature regardless of exposure to light or humidity in the range of 20-65%. Enhanced temperature associated humidity effect was responsible for the instabilities of tetracycline HCl and famotidine to different extents. Elevated temperature accelerated the degradation of all the drugs in liquid phase but light exposure was not a factor for the degradation. The degradation processes of tetracycline HCl and famotidine were highly dependent on the pH of the solution, and relatively stable profiles were achieved at pH 4.0. No potential incompatibility between the drugs under storage conditions was observed in the development of a new multi-drug delivery tablet.

Development of a rapid HPLC method for determination of famotidine in human plasma using a monolithic column.

A rapid and sensitive HPLC method using a monolithic column has been developed for quantification of famotidine in plasma. The assay enables the measurement of famotidine for therapeutic drug monitoring with a minimum detectable limit of 5 ngml(-1). The method involves simple, one-step extraction procedure and analytical recovery was complete. The separation was carried out in reversed-phase conditions using a Chromolith Performance (RP-18e, 100 mm x 4.6 mm) column with an isocratic mobile phase consisting of 0.03 M disodium hydrogen phosphate buffer-acetonitrile (93:7, v/v) adjusted to pH 6.5. The wavelength was set at 267 nm. The calibration curve was linear over the concentration range 20-400 ngml(-1). The coefficients of variation for inter-day and intra-day assay were found to be less than 8%.