High-pressure Liquid Chromatographic Method Research Articles

A Rapid Determination of Cinnarizine in Bulk and Pharmaceutical Dosage Form by LC

A simple, selective, rapid and precise reverse phase high pressure liquid chromatographic method has been developed for the estimation of cinnarizine from pharmaceutical formulation. The method was developed using MICRA‐NPS C18 (length×OD×ID =33×8.0×6.0 mm, 1.5 μm) column with a mobile phase consisting of acetonitrile, triethylamine buffer (adjusted to pH 4.5 with 10% w/v potassium hydroxide) and tetrahydrofuran in the ratio 30:66:4 respectively, at a flow rate of 0.5 mL/min. Wavelength was fixed at 253 nm. The developed method was validated for linearity, accuracy, precision, limit of detection and limit of quantitation. The proposed method can be used for the routine estimation of cinnarizine in pharmaceutical dosage form.

Plasma levels of retinoids, carotenoids and tocopherols in patients with mild obstructive sleep apnoea

OSA is associated with increased incidence of cardiovascular diseases. Pathogenic mechanisms of vascular diseases include thickened vascular walls due to the increased number of smooth muscle cells (SMC). Retinoic acid (RA) suppresses the growth of SMC, and reduced retinoid levels are associated with vascular diseases. Oxidant signalling promotes SMC growth, thus antioxidant levels may also influence the development of cardiovascular diseases. The present study tested the hypothesis that plasmas from OSA patients contain altered levels of retinoids, carotenoids and tocopherols. Plasma samples were taken before and after sleep from patients with OSA (mostly mild) without known cardiovascular diseases and from control subjects. Levels of retinoids, carotenoids and tocopherols were measured using sensitive gas chromatograph-mass spectrometry and high pressure liquid chromatography methods and total antioxidant capacity was assessed fluorometrically. Results showed that plasmas from patients with OSA had significantly lower retinyl palmitate and 9-cis RA compared with control subjects, while levels of retinol, all-trans RA and 13-cis RA were indifferent. All-transbeta-carotene and 9-cisbeta-carotene were also lower in OSA patients. Levels of all-trans RA and 13-cis RA in OSA patients were reduced after sleep compared with before sleep. OSA patients showed significantly higher delta-tocopherol compared with controls. Treatment of cultured human vascular SMC with post-sleep OSA patient plasmas promoted cell growth, but not in controls. Mild OSA exhibits altered levels of specific retinoids, carotenoids and tocopherols, which may be markers and/or mediators for the increased susceptibility of patients to vascular diseases.

Evaluation and validation of two different chromatographic methods (HPLC and LC-MS/MS) for the determination and confirmation of ochratoxin A in pig tissues

This study was undertaken to compare two different analytical methods for the determination and confirmation of ochratoxin A (OTA) in blood serum, kidney and liver of pigs. Sample clean-up was based on liquid-liquid phase extraction. The detection of OTA was accomplished with high-performance liquid chromatography (HPLC) combined either with fluorescence detection (FL) or electro spray ionization (ESI+) tandem mass spectrometry (MS–MS). Comparative method evaluation was based on the investigation of 90 samples of blood serum, kidney and liver per animal originating from different regions of Serbia. The analytical results are discussed in view of the respective method validation data and the corresponding experimental protocols. In general, analytical data obtained with (LC–MS–MS) liquid chromatography electro spray tandem mass spectro metry detection offered comparable good results at the sub-ppb concentration level. The results indicate that the liquid chromatography electro spray tandem mass spectrometric (LC-MS/MS) method was more specific and sensitive for the analysis and confirmation of ochratoxin A in pig tissues then high pressure liquid chromatography (HPLC) method after methylation of OTA.

Simultaneous analysis of neuroendocrine tumor markers by HPLC-electrochemical detection

A validated, high pressure liquid chromatographic (HPLC) method for simultaneous quantitation of urinary catecholic acids 4-hydroxy-3-methoxymandelic acid (HMMA) (vanylmandelic acid) (VMA), 4-hydroxy-3-methoxyphenylacetic acid (HVA) and 5-hydroxyindole-3-acetic acid (5-HIAA) was developed. Sample preparation involved liquid–liquid extraction of acidified urine, containing iso-HMMA (IS) as internal standard, with ether, evaporation of the organic extract, followed by reconstitution of the residue in phosphate buffer at pH 3.3. After reversed-phase HPLC at 35 °C and separation on a Licrospher 125 mm × 4 mm C 18 column (5 μm particle size) with phosphate buffer (pH 3.5)–acetone (950:50, v/v) as eluent, quantitation is achieved by electrochemical detection using coulometric detection at a potential of +350 mV. The method was successfully applied to routine diagnosis of neuroblastoma, carcinoid syndrome and pheochromocytoma.

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls—fucosylated and sialylated human lactoferrin glycopeptides—and negative controls—high mannose glycopeptides from Saccharomyces cerevisiae—that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals.

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls—fucosylated and sialylated human lactoferrin glycopeptides—and negative controls—high mannose glycopeptides from Saccharomyces cerevisiae—that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals.

Validation of an ultra high pressure liquid chromatographic method for the determination of biologically active amines in food

Biologically active amines include the so called biogenic amines, such as histamine, tyramine and cadaverine, and polyamines such as spermidine and spermine. Ultra high pressure liquid chromatography (UHPLC) is a new generation of separation techniques that takes full advantage of chromatographic principles to increase speed flow which drastically reduce analysis time. The aim of the present work was to validate a rapid method of UHPLC to detect the presence of biogenic amines and polyamines in food. Different food matrixes (wine, fish, cheese, and dry fermented sausage) were used in order to test the versatility of the method. The UHPLC method described in this article has been demonstrated as a reliable procedure to determine 12 biogenic amines and polyamines in less than 7 min of chromatographic elution. The method provides a satisfactory linearity and chromatographic sensitivity with a detection limit lower than 0.2 mg/L and a determination limit falling below 0.3 mg/L for all amines. The precision, in terms of relative standard deviation, was lower than 5% and the accuracy, as mean recovery, was between 93% and 98%, depending on the food matrix.

Phosphatidylethanol: Normalisation during detoxification, gender aspects and correlation with other biomarkers and self-reports

Aims: Phosphatidylethanol (PEth) is a direct ethanol metabolite, attracting attention as biomarker of ethanol intake. Aims of the presented studies are: 1) To further characterise the normalisation of PEth in lager samples and 2) to elucidate potential gender differences and 3) the correlation with other biomarkers and self-reports. Patients and methods: Fifty-seven alcohol dependent patients (ICD 10 F 10.25; f=9, m=48) at three study sites age 43.56 years, gamma glutamyl transpeptidase (GGT) 209.61 U/L, mean corpuscular volume (MCV) 97.35 fL, carbohydrate deficient transferrin (%CDT) 8.68, 1452.29g ethanol intake in the last 7 days (mean) were included. PEth was measured in heparinized whole blood with a high pressure liquid chromatography (HPLC) method, GGT, MCV and %CDT using routine methods. Results: PEth levels of between 0.63 and 26.95µmol/L (6.22 mean, 4.70 median, SD 4.97) at day 1 of detoxification were found. There were no false negatives at day 1. Sensitivities for the other biomarkers were 40.4% for MCV, 73.1% for GGT and 69.2% for %CDT, respectively. The correlation between alcohol intake in the last seven days prior to hospitalisation and PEth was r=.427, p<0.05. No gender differences were found for PEth levels at any time point. Conclusion: Our data suggest, that PEth is 1) a suitable intermediate term marker of ethanol intake in both sexes, 2) sensitivity is extraordinary high in alcohol dependent patients. The results add further evidence to the data that suggest that PEth has potential as a candidate for a sensitive and specific biomarker, which reflects longer lasting intake of higher amounts of alcohol and seemingly has certain advantages over traditional biomarkers.

The Value of Immunoassays for Metanephrines in the Biochemical Diagnosis of Pheochromocytomas

An undiagnosed pheochromocytoma may result in life-threatening consequences. The diagnosis of pheochromocytoma is based on the overproduction of catecholamines. Highly sensitive biochemical assays are essential to avoid false-negative results. Determinations of 24-h urinary epinephrine and norepinephrine levels are established diagnostic tools. However, they may be falsely negative in patients with a biochemically-silent or periodically-secreting pheochromocytoma. Metanephrines, which are metabolites of catecholamines, have been suggested as an alternative diagnostic tool. Urinary metanephrines are determined by high-pressure liquid chromatography (HPLC) in an increasing number of laboratories, whereas plasma metanephrines measured by HPLC are available in specialised centres only. The different HPLC methods may be cost- and time-intensive. Immunoassays such as radio- or enzyme-immunoassays may be alternative procedures. Measurement of metanephrines instead of catecholamines by either technique improved the diagnostic accuracy for the diagnosis of pheochromocytomas. Determination of plasma free metanephrines demonstrated a higher accuracy than their urinary counterparts. The use of immunoassays may be an alternative to the laborious HPLC, although the method needs to be evaluated in more detail.

Isolation and Simultaneous LC Analysis of Thiram and Its Less Toxic Transformation Product in DS Formulation

A simple and sensitive high pressure liquid chromatographic method has been developed for the simultaneous determination of Thiram and its transformation product using isocratic mixture of methanol-water (65:35) at flow rates of 0.75 mL min(-1), PDA detector using UV absorbance (lambda(max)) at 217 nm for Thiram and 265.5 nm for the transformation product. The transformation product was isolated from the commercial DS formulation and has been tentatively assigned the structure by (1)H NMR and ESI-MS spectral data. The separation is dependent on the nature of the mobile phase, its flow rate and the nature of the HPLC column. The detection limit (signal/noise; S/N = 3) for both Thiram and its transformation product was 0.2 ppm. The method has been successfully applied to analysis of soil and soybean samples spiked with Thiram, its transformation product and a commercial sample containing these products.

Detection, quantifications and pharmacokinetics of toltrazuril sulfone (Ponazuril®) in cattle

Toltrazuril sulfone (Ponazuril) is a triazine-based anti-protozoal agent with highly specific actions against apicomplexan group of organisms, which are undergoing intensive investigation. Toltrazuril sulfone may have clinical application in the treatment of Neospora. caninum and other protozoal infections in cattle. To evaluate absorption, distribution, and elimination characteristics of toltrazuril sulfone in cattle, a sensitive validated quantitative high-pressure liquid chromatography method for toltrazuril sulfone in bovine biological fluids was developed. After a single oral dose of toltrazuril sulfone at 5 mg/kg (as 150 mg/g of Marquis; Bayer HealthCare, Shawnee Mission, KS, USA), samples from six cows showed good plasma concentrations of toltrazuril sulfone, which peaked at 4821 ng/mL +/- 916 (SD) at 48 h postadministration. Thereafter, plasma concentration declined to 1950 ng/mL +/- 184 (SD) at 192 h after administration with an average plasma elimination half-life of approximately 58 h. Following oral dose of toltrazuril sulfone, the observed peak plasma concentrations were in relatively close agreement ranging from the lowest 3925 ng/mL to the highest of 6285 ng/mL with the mean peak plasma concentration being 4821 ng/mL. This study shows that toltrazuril sulfone is relatively well absorbed after oral dose in cattle. These results are therefore entirely consistent with and support the reported clinical efficacy of toltrazuril sulfone in the treatment of experimentally induced clinical cases of N. caninum and other protozoal-mediated bovine diseases.

Effective Prophylactic Treatments of Migraine Lower Plasma Glutamate Levels

The role of glutamate in migraine treatment has not been much studied, even if this amino acid seems to be crucial in the pathogenesis of migraine. Our aim was to determine if there were differences in the plasma levels of glutamate between migraine patients and control subjects and if plasma levels of glutamate in migraine patients modified after 8 weeks of prophylactic treatment. We studied 24 patients with diagnosis of migraine without aura according to International Classification of Headache Disorders, 2nd edn criteria, and 24 age- and sex-matched healthy subjects, as controls. In migraineurs the level of glutamate was measured before and after 8 weeks of prophylactic treatment (topiramate 50 mg/day, five patients; amitriptyline 20 mg/day, seven patients; flunarizine 5 mg/day, seven patients; propranolol 80 mg/day, five patients). Venous blood samples were taken in the morning, after overnight fasting, and at least 3 days after the last migraine day. Glutamate levels were measured by means of a fluorimetric detector high-pressure liquid chromatographic method. Plasma levels of glutamate were significantly higher in migraine patients-either before (61.79 +/- 18.75 micromol/l) or after prophylactic treatment (17.64 +/- 5.08 micromol/l)-than in controls (9.36 +/- 2.1 micromol/l) (P < 0.05, anova followed by Newman-Keuls' test). After prophylactic treatment, with headache frequency reduced, plasma glutamate levels were significantly lower in the same patient with respect to the prior baseline level (P < 0.0001, Student's t-test for paired data), without any differences depending on the kind of prophylactic drug. Effective prophylactic treatments reducing high glutamate plasma levels found in migraine patients could act on the underlying mechanism that contributes to cause migraine. Plasma glutamate level monitoring in migraine patients might serve as a biomarker of response to treatments and as an objective measure of disease status.

Analysis of benzo[a]pyrene metabolites formed by rat hepatic microsomes using high pressure liquid chromatography: optimization of the method.

A simple and sensitive method was developed to separate the carcinogenic polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene (BaP), and six of its oxidation metabolites generated by rat hepatic microsomes enriched with cytochrome P450 (CYP) 1A1, by high pressure liquid chromatography (HPLC). The HPLC method, using an acetonitrile/water gradient as mobile phase and UV detection, provided appropriate separation and detection of both mono- and di-hydroxylated metabolites of BaP as well as BaP diones formed by rat hepatic microsomes and the parental BaP. In this enzymatic system, 3-hydroxy BaP, 9-hydroxy BaP, BaP-4,5-dihydrodiol, BaP-7,8-dihydrodiol, BaP-9,10-dihydrodiol and BaP-dione were generated. Among them the mono-hydroxylated BaP metabolite, 3-hydroxy BaP followed by di-hydroxylated BaP products, BaP-7,8-dihydrodiol and BaP-9,10-dihydrodiol, predominated, while BaP-dione was a minor metabolite. This HPLC method will be useful for further defining the roles of the CYP1A1 enzyme with both in vitro and in vivo models in understanding its real role in activation and detoxification of BaP.

Vitamin D status assessed by a validated HPLC method: within and between variation in subjects supplemented with vitamin D3

Objective. The aim of this study was to develop and validate a high‐pressure liquid chromatography (HPLC) method for assessing vitamin D status as 25‐hydroxyvitamin D2 (S‐25OHD2) and 25‐hydroxyvitamin D3 (S‐25OHD3) in serum. Material and methods. We assessed the within‐ and between‐subject variation of vitamin D status in serum samples from four different dietary intervention studies in which subjects (n = 92) were supplemented with different doses of vitamin D3 (5–12 µg/day) and for different durations (4–20 months). Results. The HPLC method was applicable for 4.0–200 nmol S‐25OHD/L, while the within‐day and between‐days variations were 3.8 % and 5.7 %, respectively. There was a concentration‐dependent difference between results obtained by a commercial radioimmunoassay and results from the HPLC method of −5 to 20 nmol 25OHD/L in the range 10–100 nmol 25OHD/L. The between‐subject variation estimated in each of the four human intervention studies did not differ significantly (p = 0.55). Hence, the pooled standard deviation was 15.3 nmol 25OHD3/L. In the studies with 6–8 samplings during 7–20 months of supplementation, the within‐subject variation was 3.9–7.2 nmol 25OHD3/L, while vitamin D status was in the range 47–120 nmol/L. Conclusions. The validated HPLC method was applied in samples from human intervention studies in which subjects were supplemented with vitamin D3. The estimated standard deviation between and within subjects is useful in the forthcoming decision on setting limits for optimal vitamin D status.

Analysis of ochratoxin A in pig tissues using high pressure liquid chromatography (HPLC) and liquid chromatography tandem mass spectrometry (LC/MS/MS) as confirmative methods

Two different analytical methods for the determination and confirmation of ochratoxin A (OTA) in blood serum, kidney and liver of pigs have been compared. Sample clean-up was based on liquid-liquid phase extraction. The detection of OTA was accomplished with high-performance liquid chromatography (HPLC) combined either with fluorescence detection (FL) or electro spray ionization (ESI+) tandem mass spectrometry (MS-MS). Comparative method evaluation was based on the investigation of 82 samples of blood serum, kidney and liver originating from different regions of Serbia. The analytical results are discussed in view of the respective method validation data and the corresponding experimental protocols. In general, analytical data obtained with LC-MS-MS detection offered comparably good results in the sub-ppb concentration level indicating that the liquid chromatography electro spray tandem mass spectrometric (LC-MS/MS) method was more specific and sensitive for the analysis and confirmation of ochratoxin A in pig tissues than high pressure liquid chromatography (HPLC) method after methylation of OTA.

TIAMULIN RESIDUE DEPLETION IN PORK AND PIG LIVER

Tiamulin is a semi-synthetic derivate of Pleuromuti lin antibiotic; produced by Pleurotus mutilus ; active against mycoplasmas; treponemas; leptospires ; Gram positive and negative germs. The aim of the study was the study of tiamulin resi dues in pork and pig liver in order to establish th e withdrawal period. To accomplish this purpose; it w as developed a study regarding the Tiamulin residue depletion as a consequence of Tiagen 80% product administering in pigs; in a dose of 12 mg / kg / day during 5 days. After the treatment; there were slaughtered 10 anim als in the 1 st ; 3 rd ; 5 th ; 6 th and 7 th day and there were collected samples from muscles and liver. After pro cessing; these samples were analyzed by High Pressure Liquid Chromatography method (HPLC). After these determinations; it was noticed that the best representation of residual depletion in pork and pig liver is the polynomial equation and the Tiamul in concentration is lower than MRL after 6 days in pork and 5 days in liver.

High performance liquid chromatographic determination of plasma free and total tazobactam and piperacillin

A high-pressure liquid chromatography (HPLC) method with ultraviolet detection was developed for the measurement of plasma free and total tazobactam and piperacillin. This method is simple and fast, requiring only 11 min for the HPLC run and a sample preparation of about 11 min for total drugs and 10 min for free drugs. The procedure for the assay involves the treatment of plasma with acetonitrile for total drugs determination, and the use of a centrifugal filter device to deproteinize plasma for free drugs determination. The HPLC column, a Hypersil-ODS, was equilibrated with an eluent mixture composed of acetonitrile–potassium phosphate (pH 2.6). CVs for repeatability of tazobactam and piperacillin measurements ranged from 4.30 to 6.60; CVs for reproducibility ranged from 5.60 to 9.40. Mean analytical recoveries ranged from 100.4 to 103%. A linear relationship was obtained between peak area and drugs concentration in the range studied (0–62.5 mg/L for tazobactam and 0–500 mg/L for piperacillin). The equation for regression line were y = 19 x − 1.4 for tazobactam and y = 1.7 x − 0.9 for piperacillin; correlation coefficients were >0.999. The lower limit of quantitation (LLQ) for standard samples was about 0.12 mg/L for tazobactam and 0.49 mg/L for piperacillin, respectively. The lower limit of detection (LLD) was 0.06 mg/L for tazobactam and 0.24 mg/L for piperacillin. This HPLC assay for tazobactam and piperacillin is sensitive and accurate, and provides a reliable determination of both free and total tazobactam and piperacillin in human plasma, thus allowing the determination of these analytes in patients receiving tazocillin therapy.

Toxicity and bioaccumulation of the insecticide “Raid” in Wister rats

Toxicity and bioaccumulation of the insecticide "Raid" was determined to assess total animal dietary exposures in a nonoccupational environment. The study focused primarily on dietary exposure concentrations (25-960 microg/g) of the ingredients of Raid administered to rats for 10 days. Tissue concentrations of the insecticide were determined by a high-pressure liquid chromatography method, whereas established methods were used to assess the tissue levels of glucose-6-phosphate and lactic acid dehydrogenase. Results show that animal mortality progressively increased with increasing concentrations while growth (in weight) decreased. Bioaccumulation of the insecticide in the tissues was in the order of lipid > muscle > liver > brain. The indices of toxicity showed no significant effect in brain, but significant reduction of glucose-6-phosphatase and lactic acid dehydrogenase levels were observed in muscle and liver. These results suggest an inhibition of some key metabolic enzymes resulting from accumulation of the insecticide components in the tissues.

Comparison of high-pressure liquid chromatography (HPLC) and Griess reagent-spectroscopic methods for the measurement of nitrate in serum from healthy individuals in the Nordic countries

Objectives Bioavailability of NO can be estimated by measuring the concentration of nitrate (NO 3) in serum. However, the methods used for the measurement NO 3 in plasma or serum show a great degree of variation. Therefore, we compared two analytical methods for the measurement of NO 3 in serum. Design and methods The concentration of NO 3 in 600 serum samples collected from healthy individuals was determined by the HPLC and by the Griess reagent-spectroscopic method. Results The concentration of NO 3 in the samples was 29.4 ± 16.1 μmol/L and 26.2 ± 14.0 μmol/L (mean ± SD) measured by HPLC and Griess reagent-spectroscopic method respectively ( p < 0.0001). We detected a significant correlation between the two methods ( R = 0.81, p < 0.0001). Conclusions A significant correlation between the two methods may suggest that either method can be used for the measurement of NO 3 in serum, however the Griess reagent-spectroscopic method measures lower concentrations of NO 3 than the HPLC method.

Pharmacokinetic study of clazuril (Appertex®) in eggs and plasma from laying hens after single or multiple treatments, using a new HPLC method for detection

1. Anticoccidials are widely used as food additives to prevent and treat coccidiosis. They are licensed for use in a prescribed concentration and during a specific time interval with broilers and pullets, but not for laying hens. 2. This study was set up to develop a new high pressure liquid chromatography (HPLC) method to detect clazuril (CZ: (±)-2-chloro-α-(4-chlorophenyl)-4-(4,5-dihydro-3,5-dioxo-1,2,4-triazin-2(3H)-yl)-benzeneacetonitrile) in egg yolk and albumen and in plasma; to investigate both the presence of residues of CZ in eggs and its pharmacokinetic behaviour in laying hens. 3. A single oral dose (3 mg/kg BW) and multiple oral doses (3 mg/kg BW for 5 d) were investigated. The analytical method gave very good recovery (64 to 74%) in the three different matrices (yolk, albumen and plasma); precision and accuracy were within 11%. 4. After a single dose no residue was detected in eggs collected for up to 10 d, while following multiple dose treatment, CZ residues were detected until 10 d after the end of treatment. The concentration of the drug was higher in yolk than in albumen with a maximum ratio of 10 : 1. 5. Pharmacokinetics of CZ in laying hens after a single dose showed a detectable concentration of the drug up to 24 h. It reached a steady state after the third administration in multiple dosing. 6. Although further studies are necessary, these results indicate that a single oral dose of CZ could be used as an anticoccidial for laying hens due to the lack of residues in eggs.