Validation and robustness testing of a HPLC method for the determination of avermectins and moxidectin in animal liver samples using an alumina column clean-up

A multi-residue method has been developed for the quantitative determination of moxidectin, abamectin, doramectin and ivermectin in liver samples, with capability for qualitative identification of the presence of eprinomectin. Liver samples are extracted with isooctane, followed by clean-up on alumina-N solid phase extraction (SPE) cartridges. Extracts are derivatised and determined by high-performance liquid chromatography (HPLC) with fluorescence detection. The method was validated using bovine liver fortified at levels of 4 and 20 micrograms kg-1 with the drugs. The mean recovery from bovine liver ranged between 90 and 96%. The intra and inter-assay variations showed RSD typically of < 5% and < 10%, respectively. The procedure was applied also to ovine and porcine liver, giving similar results. A robustness study, carried out on the alumina clean-up step, indicated that the step is relatively insensitive to method changes. However, significant differences overall were found for the type of alumina and/or commercial SPE cartridge used. The limit of quantitation of the method is 2 micrograms kg-1 (ppb).     

Development and optimisation of an improved derivatisation procedure for the determination of avermectins and milbemycins in bovine liver

A robust procedure has been developed to overcome the instability problems experienced with the fluorescent derivative of eprinomectin. The procedure involves addition of acetic acid, together with the typical reagents methylimidazole and trifluoroacetic anhydride, to produce a fluorescent molecule that can be determined by high performance liquid chromatography (HPLC) with fluorescence detection. Derivatisation is completed in 30 min at 65 degrees C. This derivatisation procedure was shown to be suitable, also, for the related compounds, moxidectin, abamectin, doramectin and ivermectin. A multi-residue method for these compounds in bovine liver has been developed using the derivatisation procedure. Samples are extracted with acetonitrile; followed by clean-up on deactivated alumina and C18 solid phase extraction (SPE) cartridges. The method was validated using bovine liver fortified at levels of 4 and 20 micrograms kg-1 with the drugs. The mean recovery ranged between 73 and 97%. The intra- and inter-assay variations showed relative standard deviations typically of < 6% and < 14%, respectively. The limit of quantitation of the method is 2 micrograms kg-1 (ppb).     

High Performance Liquid Chromatographic Determination of Emetine in Corn

Abstract

A High performance liquid chromatographic (HPLC) method for the quantitative analysis of emetine in corn is described. Corn kernels are removed from the ears, blended, and extracted with methanol. The extract is filtered through a 0.45 um Acrodisc filter and analyzed by HPLC using a LC-18-DB column. For detection, a fluorescence detector set at excitation of 285 and emission at 316 nm is used. The recoveries of samples fortified between 0.1 ppm to 20 ppm with emetine ranged from 95.5% to 103.3%.     

Multi-residue analysis of avermectins and moxidectin by ion-trap LC-MSn

A multi-residue method was developed and validated for the quantitation and confirmation of avermectins and moxidectin residues in bovine liver. Target analytes were extracted from liver homogenate using C8 solid phase cartridges, chromatographed under basic pH conditions in order to promote the formation of analyte anions, and detected by ion-trap mass spectrometry (MS) in negative ion mode using an atmospheric pressure chemical ionization interface (APCI). The method provided detection capabilities (CC beta, where beta = 0.05) for eprinomectin, abamectin, doramectin, moxidectin and ivermectin of 3.1, 3.2, 2.2, 4.0 and 3.2 ng g-1 liver respectively, well below their respective maximum residue limits (MRLs). The critical concentrations for MRL compliance (CC alpha, where alpha = 0.01) were 840, 28, 130, 130 and 130 ng g-1 respectively. Analysis of liver fortified at the appropriate MRLs gave recoveries (% +/- RSD) of 70.9 +/- 11.6 (n = 14), 69.1 +/- 3.9 (n = 13), 65.9 +/- 6.4 (n = 19), 69.7 +/- 9.3 (n = 19) and 73.2 +/- 10.5 (n = 19), respectively, for each analyte. Calibration curves fitted a second order polynomial function (R2 > or = 0.9978) over a wide range of concentrations (0 to 10,000 ng ml-1). The detection of two daughter-ions for each analyte allowed for quantitation and the confirmation of identity. The method is suitable for application in European Union statutory veterinary drug residue surveillance programmes, since it fulfills appropriate analytical criteria, and has the particular advantage of enabling high throughput multi-residue quantitation and confirmation of the target analytes.     

Determination of abamectin, doramectin, emamectin, eprinomectin, ivermectin, and moxidectin in milk by liquid chromatography electrospray tandem mass specrometry

The avermectin and milbemycin families of compounds are derived from naturally occurring yeasts. They have proven to be potent preventatives against a variety of pests such as insects and parasites. Only eprinomectin and moxidectin are currently approved for use on lactating cattle with tolerances in milk of 12 microg/kg for eprinomectin and 40 microg/kg for moxidectin. Detection of misuse or inadvertent contamination in milk requires a sensitive and definitive analytical method. A method has been developed for the determination of 5 avermectins and 1 milbemycin in milk using a simple liquid-liquid extraction and liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis. Ivermectin (IVR), doramectin (DOR), abamectin (ABA), eprinomectin (EPR), emamectin (EMA), and moxidectin (MOX) were extracted from whole milk by partitioning into acetonitrile with a subsequent solvent exchange into methanol-water. Simultaneous confirmation and quantification were achieved with LC separation, positive electrospray ionization (ESI+), and MS/MS. The limits of detection ranged from 16 pg/g (ppt) for EMA to 1.7 microg/g (ppb) for MOX.     

改良的QuEChERS结合高效液相色谱-串联质谱同时测定水产品中7种阿维菌素类药物残留

建立了改良的QuEChERS结合高效液相色谱-串联质谱(HPLC-MS/MS)同时测定水产品中7种阿维菌素类药物(阿维菌素、伊维菌素、多拉菌素、塞拉菌素、乙酰氨基阿维菌素、莫西菌素和埃玛菌素)的分析方法。样品经0.2%(v/v)氨化乙腈提取,3 g无水硫酸镁和2 g无水硫酸钠除水剂和沉淀蛋白质,100 mg十八烷基硅烷(C18)和500 mg无水硫酸镁净化。以0.1%(v/v)甲酸乙腈(含5 mmol/L乙酸铵)-0.1%(v/v)甲酸水溶液(含5 mmol/L乙酸铵)为流动相,采用Varian Pursuit ULTRA C8色谱柱(100 mm×2.0 mm,2.8μm)进行分离。在加热电喷雾离子(HESI)源、正离子模式下采用多反应监测模式检测,基质匹配标准曲线外标法定量。阿维菌素、伊维菌素、多拉菌素、塞拉菌素、乙酰氨基阿维菌素和莫西菌素在2~200μg/L范围内、埃玛菌素在0.2~20μg/L范围内呈线性相关,相关系数(r)均≥0.997 2,水产品中阿维菌素类药物的加标回收率为71.6%~112.8%,相对标准偏差(RSD)为4.7%~13.1%,不同水产品的基质效应均小于15%。阿维菌素、伊维菌素、多拉菌素、塞拉菌素、乙酰氨基阿维菌素和莫西菌素的定量限均为5μg/kg,埃玛菌素的定量限为0.25μg/kg。该法操作简便,重复性好,适用于水产品中7种阿维菌素类药物残留量的同时测定。     

二乙酰酒石酸酐柱前手性衍生化反相高效液相色谱法拆分心得安对映体

以（Ｒ，Ｒ）－Ｏ，Ｏ－二乙酰基酒石酸酐作柱前手性衍生化试剂，用ＯＤＳ柱拆分了心得安对映体，探讨了衍生化反应条件，研究了ＰＨ及流动相组成对色谱保留行为的影响，发现心得安的两种非对映异构体衍生物的荧光响应有显著差别，对心得安对映体的半制备色谱拆分规律作了新的探讨，确定用Ｓｈｉｍ－ｐａｃｋＣＬＣ－ＯＤＳ柱进行半制备色谱拆分的最佳条件，得到的光学异构体纯度在９９％以上。     

高效液相色谱－荧光检测法测定人血浆中匹伐他汀的含量

 Pitavastatin belongs to the class of coenzyme A reductase inhibitors. Very few methods of assaying pitavastatin from human plasma are available in literature. An analytical method is presented for the determination of the drug from human plasma making use of the fluorescent property of the drug. The drug is extracted from plasma using ethyl acetate under neutral condition and then analyzed by reversed-phase high performance liquid chromatography (HPLC) with fluorescence detection (λEx 245 nm; λEm 420 nm). Analysis of pitavastatin was carried out on a C18 HPLC column using a gradient flow of mobile phase (0.01 mol/L monobasic potassium phosphate (pH 3.20)-acetonitrile, 63∶37, v/v). Fluorescein isothiocyanate was used as internal standard. The dynamic range of assay was 3 to 50 ng/mL. The intraday precision was less than 10% and accuracy ranged from 95.2% to 112.6%. The same for interday check was less than 12% and 92.8% to 105.1%, respectively. The drug was found to be stable under the assay conditions. The developed method is simple, precise, accurate, and stable. This indicates that it can be applied to routine analysis of this drug in human subjects where there are large numbers of samples without the need of specialized instruments like column switching.     

Semi-Preparative High Performance Liquid Chromatographic Separation of Carbon-14 Labeled Avermectin B1a from a Mixture of Avermectins

Abstract

A semi-preparative high performance liquid chromatographic method has been developed to separate carbon-14 labeled avermectin B_1a from a fermentation mixture of carbon-14 labeled avermectins, i. e., avermectins A₁a, A₁b, A₂a, A₂b, B₁a, B₁b, B₂a, and B₂b. Two HPLC systems were employed for the separation: I. A Whatman M20, Partisil 10, normal phase column and a solvent system of 10% ethanol in isooctane (v/v), and II. A Whatman M20, Partisil 10, ODS-3, reverse phase column and a solvent system of acetonitrile/methanol/water (56:18:26, v/v/v); the flow rate was 18 ml/ min. Avermectin separations were monitored using ultraviolet detection (254 nm). Further analyses of avermectin B₁a were done using analytical HPLC and TLC/radioassay to check compound purity and identity.     

Determination of five 4-hydroxycoumarin rodenticides in animal liver tissues by ion chromatography with fluorescence detection

A novel analytical method is proposed for rapid simultaneous determination of five 4-hydroxycoumarin rodenticides in animal liver tissues by eluent generator reagent free ion chromatography (RFIC) with fluorescence detection. Rodenticides were initially extracted from homogenized animal liver tissues with ethyl acetate and the extracts subjected to a solid-phase extraction process using Oasis HLB cartridges. The IC separation was carried out on an IonPac AS11 analytical column (250 mm x 4.0 mm) using gradient KOH containing 10% acetonitrile as organic modifier at a constant flow rate of 1.0 mL/min. The analytes were detected by fluorescence at an excitation wavelength of 270 nm and an emission wavelength of 380 nm. The average recoveries of the objective compounds spiked in animal liver tissues were between 81% and 98%. The limits of quantification (LOQs) were 0.004-0.010 mg/kg for them. Within-day and day-to-day relative standard deviations (RSD) were less than 8.5% and 9.7%, respectively. It was confirmed that this method could be used in a toxicological analysis.     

Determination of sodium monofluoroacetate (1080) in biological samples as its 4-bromomethyl-7-methoxycoumarin derivative by RP-HPLC

A high-performance liquid chromatography (HPLC) method with fluorescence detection is described for the determination of sodium monofluoroacetate (MFA-Na) in biological samples. 4-Bromomethyl-7-methoxycoumarin is used as a derivatization reagent and reacted with MFA-Na to form 7-methoxy-4-methylenecoumarin monofluoroacetate for HPLC analysis. Chromatographic separation is performed on a Hewlett Packard RP-18 column using methanol-water (60:40, v/v) as the mobile phase. A fluorescent detector is employed with the excitation and emission wavelengths as 319 nm and 390 nm, respectively. The novel method yields a good linear relationship when the concentration of MFA-Na is within 1 and 500 nmol/mL (r = 0.9996). The detection limit is 50 pmol/mL. The established method is applied to determine MFA-Na in biological samples. The recovery rates of MFA-Na are between 81% and 88%, and the relative standard deviations are less than 5%. The method shows good sensitivity and selectivity for the determination of MFA-Na in biological samples.     

超高效液相色谱-电喷雾电离串联质谱联用法检测茶叶中阿维菌素类药物残留

建立了采用超高效液相色谱-电喷雾电离串联质谱同时检测茶叶中爱比菌素、甲胺基阿维菌素、乙酰胺基阿维菌素、伊维菌素、多拉菌素、莫西丁克残留的方法。试样经饱和氯化钠溶液浸润后,用乙腈提取,C18固相萃取小柱净化,超高效液相色谱-电喷雾串联质谱法(UPLC/ESI-MS/MS)测定。对流动相、监测离子、校正曲线等进行了优化和探讨。6种分析物在2.0～50 μg/L范围内线性关系良好,相关系数均大于0.9920。莫西丁克在5,10,20 μg/kg,其余分析物在2,5,10 μg/kg加标水平的平均回收率为61.7%～85.4%,相对标准偏差为9.37%～17.19%。该方法可靠、稳定,可满足茶叶中阿维菌素类药物残留检测与确证的需要。     

Determination of urinary free cortisol by high performance liquid chromatography with sulphuric acid-ethanol derivatization and column switching.

A rapid and highly sensitive determination method for urinary free cortisol has been developed using reversed phase high performance liquid chromatography (HPLC) with a precolumn for sulphuric acid-ethanol fluorescence derivatization and column switching. Urinary cortisol, eluted from the octadecylsilane-bonded silica (ODS) minicolumn with 90% aqueous ethanol, was derivatized with the addition of sulphuric acid only at ambient temperature. Cortisol derivatives injected directly onto the ODS precolumn were purified on-line. After switching the columns, the cortisol derivative was separated on an ODS analytical column with a retention time of 15.3 min and monitored at an emission wavelength of 520 nm (exitation wavelength of 365 nm) to decrease the detection limit to 0.26 microgram/dL (signal-to-noise ratio = 3). The automated HPLC operation resulted in good reproducibility and recovery of the stable cortisol derivative at 5 degrees C.     

Improved high-performance liquid chromatographic procedure for the determination of lasalocid in chicken tissues and egg using polymeric and porous graphitic carbon columns.

A high-performance liquid chromatographic (HPLC) method for the determination of the ionophore coccidiostat lasalocid in poultry muscle and eggs was developed. The drug was extracted from tissue with acetonitrile. The extract was partitioned between saturated salt and carbon tetrachloride and the organic layer evaporated to dryness. Clean-up was by solid-phase extraction on a silica column. HPLC analysis was carried out on either a polymeric PLRP-S or a porous graphitic carbon Hypercarb column with a basic mobile phase and fluorescence detection with excitation at 310 nm and emission at 420-430 nm. Average recoveries from poultry muscle at the 0.002, 0.010 and 0.050 mg kg-1 levels were 65.7, 72.0 and 77.9%, respectively. Average recoveries from egg at the 0.010 and 0.100 mg kg-1 levels were 76.2 and 76.4%, respectively.     

Determination of avermectins in commercial formulations using microemulsion electrokinetic chromatography

Microemulsion electrokinetic chromatography (MEEKC) was developed for quantitative analysis of avermectins, such as abamectin, doramectin and ivermectin, in commercial formulations, using the microemulsion buffer containing a 50 mM phosphate buffer at pH 2.5, 1.1% (v/v) n-octane as oil droplets, 180 mM sodium dodecylsulphate as surfactant, 890 mM 1-butanol as co-surfactant and 30% (v/v) ethanol as organic co-solvent. High accuracy and precision of the method were obtained. The contents of avermectins in commercial formulations determined by MEEKC were found to be insignificantly different with those determined by high performance liquid chromatography (HPLC). Therefore, MEEKC can be used an alternative method to HPLC for quantitative determination of avermectins.     

Development and Validation of High Performance Liquid Chromatographic and UV-Derivative Spectrophotometric Methods for the Determination of Sotalol Hydrochloride in Tablets

Abstract

High performance liquid chromatographic (HPLC) and UV derivative spectrophotometric (UVDS) methods were developed and validated for the quantitative determination of sotalol hydrochloride in tablets. The HPLC method was performed on a C18 column with fluorescence detection. The excitation and emission wavelengths were 235 and 310 nm, respectively. The mobile phase was composed of acetonitrile-water containing 0.1% trietylamine (7:93 v/v) and pH adjusted to 4.6 with formic acid. The UVDS method was performed taking a signal at 239.1 nm in the first derivative. The correlation coefficients (r) obtained were 0.9998 and 0.9997 for HPLC and UVDS methods, respectively. The proposed methods are simple and adaptable to routine analysis.     

A high-performance liquid chromatographic method for the determination of monofluoroacetate

A simple isocratic high-performance liquid chromatographic (HPLC) method for the quantitative analysis of monofluoroacetic acid (MFA), the toxic substance of Dichapetalum cymosum, in plant material, rumen contents (gastric contents), and liver samples is described. A suitable HPLC column that gives optimum sensitivity, accuracy, precision, and separation of MFA is identified. A C-610 organic acid analysis column at ambient temperature with 0.02M H3PO4 as an eluent and ultraviolet detection at 210 nm is utilized to quantitate MFA. Using this method, the average percentage recovery in plant material, bovine liver, and rumen samples is 94.8%, and a detection limit of 12 microg/L is achievable.     

Determination of femtomole/milliliter concentrations of enprostil acid in human plasma using high-performance liquid chromatography-laser-induced fluorescence detection

This paper describes the use of multiple-column high-performance liquid chromatography (HPLC) combined with laser-induced fluorescence for the determination of femtomole/milliliter concentrations of enprostil acid, a prostaglandin analogue, in human plasma. The drug is isolated from plasma by phenyl solid-phase extraction and fluorescently labeled at its carboxyl functional group with a large excess of 2-bromoacetyl-6-methoxynaphthalene. A multi-column method using both normal- and reversed-phase chromatography is necessary to separate the labeled drug from the unreacted reagent. Post-column dilution of the mobile phase with water after the reversed-phase chromatography allows on-line concentration of the labeled analyte onto a guard column prior to the microbore HPLC. A loop guard column device provides a simple way to inject up to 1.0 ml of sample solution onto a microbore column without significantly reducing the column efficiency. A 325-nm He-Cd laser is used to excite the labeled drug, and fluorescence emission is monitored at 450 nm. Using this system, we are able to derivatize, detect, and quantify 5 pg of the prostaglandin analogue in 1.0 ml of plasma.     

A sensitive and selective determination method of histamine by HPLC with intramolecular excimer-forming derivatization and fluorescence detection

A highly sensitive, selective and simple method is described for the determination of histamine by high-performance liquid chromatography (HPLC) with fluorescence detection. The method is based on an intramolecular excimer-forming fluorescence derivatization of histamine with 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), followed by reversed-phase HPLC. Histamine, having two amino moieties in a molecule, was converted to the dipyrene-labeled derivative by reaction with PSE. The derivative afforded intramolecular excimer fluorescence (450-540 nm), which can clearly be discriminated from the monomer fluorescence (370-420 nm) emitted from PSE. Typically, a 10 micro L sample solution was mixed with 100 micro L of derivatization reagent solution, which was a mixture of 0.5 mm PSE in acetonitrile and 0.5 mm potassium carbonate in water (8:2, v/v). The derivatization was carried out at 100 degrees C for 90 min. The PSE derivative of histamine could be separated by reversed-phase ODS column with isocratic elution using acetonitrile:water (82:18, v/v) containing 0.03% triethylamine. The detection limit (singnal-to-noise ratio = 3) of histamine was 0.5 fmol for a 30 micro L injection. The method was successfully applied to the determination of histamine in human urine, and had enough selectivity and sensitivity for urinary histamine quantification.     

Determination of vitamin B12 in multivitamin tablets and fermentation medium by high-performance liquid chromatography with fluorescence detection

A novel method for the determination of vitamin B12 by high-performance liquid chromatography with fluorescence detection is reported. The method was simple and highly sensitive with good precision. Vitamin B12 was analyzed by HPLC on a muBondapak C18 column (300x3.9 mm, 10 microm) with methanol-water (30:70) as mobile phase and fluorescence detection at 305 nm (with excitation at 275 nm). The calibration graph was linear from 1.000 to 100.0 ng ml(-1) for vitamin B12 with a correlation coefficient of 0.998 (n=6). The detection limit was 0.1 ng ml(-1). The method was successfully applied to the determination of vitamin B12 in vitamin B12 tablets, multivitamin tablets and fermentation medium. The recovery was from 94 to 102% and the relative standard deviation was in the range of 1.8 to 4.1%.