Improvement of chemical analysis of antibiotics. X. Determination of eight tetracyclines using thin-layer and high-performance liquid chromatography

Analytical methods for eight tetracyclines (TCs) were established using silica gel high-performance thin-layer chromatography (HPTLC), reversed-phase thin-layer chromatography (RP-TLC) and high-performance liquid chromatography (HPLC). Good separations of eight TCs were obtained using chloroform-methanol-5% disodium ethylenediaminetetraacetate solution (65:20:5) (lower layer) and methanol acetonitrile 0.5 M oxalic acid solution (1:1:4) (pH 3.0) on silica gel HPTLC and C8 TLC plates, respectively. A combination of HPTLC and RP-TLC made possible the identification of the eight TCs. Each calibration graph was linear between 0.1 and 1.0 microgram using UV densitometry except for rolitetracycline. For detection reagents, the diazonium salts including Fast Violet B gave variously coloured spots with the eight TCs and good sensitivities were obtained except with minocycline. In HPLC, the simultaneous analysis of the eight TCs on a C8 column was possible using methanol-acetonitrile-0.01 M oxalic acid solution (1:1.5:7) adjusted to pH 3.0 as the mobile phase. A linear relationship was obtained between 1.0 and 10 ng using the usual sample preparation except for rolitetracycline. The direct determination of rolitetracycline was possible using tetrahydrofuran, dimethyl sulphoxide and the mobile phase as solvents for preparation of the sample. For the determination of residual rolitetracycline, it was effective to measure the amount of rolitetracycline as tetracycline by HPLC, HPTLC and RP-TLC after conversion of rolitetracycline to tetracycline by incubating for 5 min in methanol at 50 degrees C.     

Fast determination of tetracycline antibiotics in different media by high-performance liquid chromatography

Summary The use of high-performance liquid chromatography (HPLC) for the identification and determination of tetracycline antibiotics is reviewed. HPLC chromatograms provide fast identification by retention time, t_R, and precise quantitation by measurement of peak height or peak area. For separation of tetracycline compounds, most HPLC methods use reversed-phase C₁₈ or C₈ columns and UV detection. The HPLC solvent system should have a pH of about 6 to prevent steric changes in the tetracycline molecule. For accurate quantitation it is necessary to avoid tailing and this is accomplished by adding a zwitter ion to the solvent system. Methanol and acetonitrile are frequently used as organic modifiers in these solvent systems. In a single analysis, HPLC methods can be used to separate as many as nine or ten commercially used tetracycline compounds and to determine four to five tetracyclines in commercial tetracycline preparations or in biological fluids.     

Analyses of tetracyline antibiotics by reversed-phase high-performance liquid chromatography

Summary A high-performance liquid chromatographic (HPLC) separation was developed that is generally applicable to nine commercially important tetracyline antibiotics. The general method uses an isocratic system and mobile phase consisting of 0.001MEDTA, pH 6.6, and methanol and a Vydac C₁₈ reversed-phase column. Quantitation of the particular tetracyline in some commercial preparations is accomplished by adjusting the mobile phase composition. Quantitative assays were developed for small amounts of 4-epitetracycline in tetracycline (TC) preparations, demeclocycline in minocycline preparations and TC in chlortetracycline (chlor-TC) preparations. A fast HPLC assay for potency was also developed for chlor-TC and minocycline in these commercial preparations.     

Utilization of a scientifically operated charge-coupled device detector for high-performance thin-layer chromatographic analysis of tetracyclines

A high-performance thin-layer chromatography (HPTLC) method using a scientifically operated charge-coupled device detector is described for the assay of tetracycline pharmaceutical products. Quantitative information can be obtained for all samples on a TLC plate within a few seconds. The separation efficiency and detection limits were determined on both normal phase and reverse phase TLC plates. Fluorescence detection mode offers higher sensitivity than fluorescence quenching mode. The dynamic range, sensitivity, accuracy and precision of the system were evaluated. Detection limits of the impurities are in the range of 0.1 to 0.5 ng or 0.3 to 1% of tetracycline, depending on the compound, with a recovery percentage over 85%. The existing impurities in tetracycline capsules were determined using both HPLC and HPTLC techniques. All of the impurities were below the regulation level.     

HPLC technique for quantitation of Chimassorb 944, and its evaluation in analysis of real and standard samples of polyolefins

A high performance liquid chromatography (HPLC) method is presented for the determination of Chimassorb 944 in polymeric matrix. A reversed phase column octadecyl silane (ODS) is used as a stationary phase. As a mobile phase, a mixture of THF:methanol:triethanol amine (90:10:1.5) (v:v:w) is used. The HPLC system was equipped with an UV detector and the absorbance of the analyte was recorded at 240 nm. In the case of polymers, 0.5 g of them along with 100 mg Irganox 1010 (for preventing oxidation of Chimassorb 944) were dissolved in boiling xylene, and then extracted with sulfuric acid 1 M four times. The extract was neutralized with sodium hydroxide solution and the analyte was re-extracted into carbon tetrachloride and then injected to the HPLC system. This method is an accurate and relatively fast technique for determination of Chimassorb 944 in polymers.     

HPLC separation of tetracycline analogues: comparison study of laser-based polarimetric detection with UV detection

A sensitive and specific high-performance liquid chromatography (HPLC) method based upon laser-based polarimetric detection is developed for the determination of six tetracycline analogues. By interfacing the laser-based polarimeter online with an HPLC system, the specific rotation of each analogue is obtained as compounds elute from the separation system. The six structurally similar tetracycline analogues exhibit significant differences in specific rotations. The experiments suggest that specific rotation can be useful in identifying closely related tetracycline analogues. Linear relationships are found to be in the range of 0.342-0.0043 mg for the tetracycline analogues. Five of the six analogues exhibit excellent linearity (R(2) value >/= 0.99). The polarimetric results are compared with UV detection. The HPLC-laser-based polarimetric detection instrument is able to quantitate the studied tetracycline analogues with high precision, accuracy, and sensitivity, which make it useful for the development of a standard method for the determination of tetracyclines in biological specimens. The performance of the HPLC-polarimetric system for the analysis of tetracyclines in a biological matrix is evaluated. The selectivity of polarimetric detection provides a distinct advantage in the analysis of tetracycline analogues in milk. The HPLC-polarimetric system provides a rapid and sensitive technique that involves minimal sample cleanup and pretreatment for the analysis of tetracyclines in milk.     

Determination of RS,E/Z-tocotrienols by HPLC

Synthetic alpha-tocotrienol was separated into four geometrical E/Z side chain isomers by preparative HPLC (permethylated beta-cyclodextrin phase). The isolated isomers were resolved in ethylene glycol dimethyl ether, converted into the corresponding methyl ether using dimethyl sulfate, and the tocotrienol methyl ethers were extracted with n-hexane. A subsequent HPLC separation on a chiral phase (adsorbent cellulose derivated with 3,5-dimethyl phenyl carbamate) discriminates between the enantiomers of each E/Z side chain isomer, achieving the complete resolution of the eight occurring synthetic RS,E/Z-alpha-tocotrienols. The method can be shortened by omitting the preparative separation of the E/Z tocotrienol isomers prior to the chromatography on the chiral dimethyl phenyl carbamate phase. The simplified method achieved the following separation: RS,E/Z-alpha-tocotrienol separated into five peaks, RS,E/Z-beta-tocotrienol into eight, RS,E/Z-gamma-tocotrienol into six and RS,E/Z-delta-tocotrienol into eight peaks. The naturally occurring R,E-E-tocotrienol isomer could be identified within the synthetic RS,E/Z-isomers by co-chromatography with tocotrienol methyl ethers derived from natural sources, respectively.     

Use of Alkaline Degradation for the Analysis of Dihydrotriazines via High Pressure Liquid Chromatography

Abstract

An HPLC method is described for the determination of 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(3′,4′-dichlorobenzyloxy)-1,3,5-triazine hydrochloride (WR 38839). The procedure required the isomerization of the drug sample by alkaline treatment with sodium hydroxide, as the parent compound was retained by the column. The reaction product of the drug was analyzed by HPLC using a strong cation exchange resin as the stationary phase and glycine buffer, pH 10.4 as the mobile phase. The product was isolated and identified by TLC, UV, IR, mass spectroscopy and elemental analysis. The postulated mechanism indicates that this would be a general analytical method for dihydrotriazine compounds. This technique, developed for the assay of the dihydrotriazine in an aqueous system, was successfully applied to rat urine samples spiked with the drug.     

Ruffling-induced chirality: synthesis, metalation, and optical resolution of highly nonplanar, cyclic, benzimidazole-based ligands

Expedient five-step syntheses of a cyclic bis(benzimidazole)-based amide 5 and two sterically more hindered analogues 23-24 have been developed. These amides are chiral due to the inherent ruffling of the macrocyclic plane. Racemization of the optical antipodes of these compounds has been studied using dynamic chiral stationary phase HPLC. These studies reveal that, while the parent amide 5 racemizes rapidly, for the sterically more hindered amides 23-24, the rate of racemization is significantly reduced. Bis(benzimidazole)-based amides 5 and 23-24 form stable Ni(II) complexes 25-27, respectively. Like their parent ligands, complexes 25-27 are chiral due to their highly ruffled geometry. Studies of these complexes by chiral stationary phase HPLC reveal that metalation leads to a much lower rate of racemization. Incorporation of a strap can slow racemization even further. A series of strapped cyclic amides 54-57, along with their corresponding dimers 58-61, have been prepared. The rate of racemization for amides 54-57 is strongly dependent on the length of the strap. X-ray single-crystal structure analysis of the Ni(II) complex of strapped amide 54 reveals that the bis(benzimidazole) core retains its highly ruffled shape, with the two phenyl rings of the macrocycle located anti to the strap. Chiral separation of strapped ligands 54-57 and their corresponding Ni(II) complexes is shown to be facile by chiral stationary phase HPLC.     

Determination of sulfonamides and trimethoprim using high temperature HPLC with simultaneous temperature and solvent gradient

A fast HPLC method for the analysis of eight selected sulfonamides (SA) and trimethoprim has been developed with the use of high temperature HPLC. The separation could be achieved in less than 1.5 min on a 50 mm sub 2 microm column with simultaneous solvent and temperature gradient programming. Due to the lower viscosity of the mobile phase and the increased mass transfer at higher temperatures, the separation could be performed on a conventional HPLC system obtaining peak widths at half height between 0.6 and 1.3 s.     

Improvement of chemical analysis of antibiotics. V. A simple method for the analysis of tetracyclines using reversed-phase high-performance liquid chromatography

An isocratic high-performance liquid chromatographic method for the determination of tetracyclines is described using a mobile phase containing oxalic acid and C8- and C18-modified silica gel columns. For good separations of tetracyclines, oxalic acid concentrations of above 0.01 and 0.2 M respectively for parent tetracyclines (group I) and impurities in tetracycline (group II) are required. The optimum pH of the aqueous oxalic acids solution in the mobile phase is 2.0. The combinations of the C8-modified silica gel column with methanol-acetonitrile-0.01 M aqueous oxalic acid solution pH 2.0 (1:1.5:5) and the C18-modified silica gel column with methanol-acetonitrile-0.2 M aqueous oxalic acid solution pH 2.0(1:1:3.5) gave satisfactory results for groups I and II, respectively.     

Development and validation of an HPLC method for tetracycline‐related USP monographs

A novel reversed‐phase HPLC method was developed and validated for the assay of tetracycline hydrochloride and the limit of 4‐epianhydrotetracycline hydrochloride impurity in tetracycline hydrochloride commercial bulk and pharmaceutical products. The method employed L1 (3 µm, 150 × 4.6 mm) columns, a mobile phase of 0.1% phosphoric acid and acetonitrile at a flow rate of 1.0 mL/min, and detection at 280 nm. The separation was performed in HPLC gradient mode. Forced degradation studies showed that tetracycline eluted as a spectrally pure peak and was well resolved from its degradation products. The fast degradation of tetracycline hydrochloride and 4‐epianhydrotetracycline hydrochloride in solution was retarded by controlling the autosampler temperature at 4 °C and using 0.1% H₃PO₄ as diluent. The robustness of the method was tested starting with the maximum variations allowed in the US Pharmacopeia (USP) general chapter Chromatography <621>. The method was linear over the range 80–120% of the assay concentration (0.1 mg/mL) for tetracycline hydrochloride and 50–150% of the acceptance criteria specified in the individual USP monographs for 4‐epianhydrotetracycline hydrochloride. The limit of quantification for 4‐epianhydrotetracycline hydrochloride was 0.1 µg/mL, 20 times lower than the acceptance criteria. The method was specific, precise, accurate and robust. Copyright © 2014 John Wiley & Sons, Ltd.     

Validation of an HPLC method for the determination of *-dichlorobenzene and naphthalene in mothrepellents

 The determination of dichlorobenzene and naphthalene in commercial repellents used in Spain has been validated. This was done using an isocratic regime, to test the reverse -phase HPLC system with acetonitrile: water 65 : 35 (v: v) as the mobile phase, at 20  °C. This technique is proposed for the modular validation of the HPLC system . The results obtained with this method show good agreement with the results provided by the manufacturers of the mothrepellents. Received: 21 December 1998 / Accepted: 4 May 1999     

High Performance Liquid Chromatographic Analysis of Tolmetin,Indomethacin and Sulindac in Plasma

Abstract

Isocratic and gradient reversed phase high-performance liquid chromatographic (HPLC) methods for the quantitation of tolmetin, indomethacin, and sulindac and their respective metabolites in plasma were developed. Only the determination of the parent drugs was possible using the isocratic technique. Specific, simultaneous determination of each drug and its respective metabolites was achieved using the gradient technique. The effect of pH and ionic concentration of the mobile phase on retention time was studied. Statistical analysis demonstrated excellent precision and linearity over the following ranges: 1–40, 0.1–3, and 0.1–3 ug/ml plasma for tolmetin, indomethacin, and sulindac respectively. Both methods have been applied to the analysis of patient samples.     

Determination of tetracyclines in ovine milk by high-performance liquid chromatography with a coulometric electrode array system

A method has been developed to analyze residual tetracyclines (TCs) (oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC), methacycline (MTC), doxycycline (DC)) in ovine milk, using high-performance liquid chromatography (HPLC) with a coulometric electrode array system. The samples were pretreated, using liquid-liquid extraction based on hexane. The chromatography was performed, using a C18 column (150 mm x 4 mm i.d. and 5 microm) with a mobile phase: sodium phosphate monobasic dihydrate (pH 2.2, 0.05 M)-acetonitrile (78:22, v/v). The flow rate of mobile phase was kept constantly at 1ml/min. The residues were monitored by an ESA electrochemical detector. Potentials of four electrodes in series were set at 400, 660, 680 and 700 mV, respectively. The first electrode was set to remove those interfering substances that may co-elute with TCs and the other three electrodes were used for quantification. The maximal potential of our detection was 700 mV. Calibration curve showed good linearity and the detection limit of TCs was 12.5, 20, 25, 10 and 25 ng/ml, respectively. Optimization of the pH of the mobile phase, the proportion of acetonitrile and the pH of the pretreatment were also performed. Recoveries of TCs from spiked samples were more than 88% and the relative standard deviations were less than 4.3%. This method was reliable, sensitive, economical and suited for routine monitoring of TC residues in ovine dairy milk.     

Diastereospecific HPLC Analysis of the Antiulcer Drug,Enprostil, in a Soft Elastic Gelatin Capsule Formulation

Abstract

Enprostil, an E-type prostaglandin analog, is formulated as a 0.3 mM propylene carbonate solution filled into soft elastic gelatin capsules. A characteristic structural feature of enprostil is the presence of two unresolved chiral centers, yielding a drug molecule that exists as two diastereomeric pairs of enantiomers. This report describes an HPLC method for quantitatively determining the relative amounts of enprostil diastereomers in soft elastic gelatin capsules. Using a Spherisorb ODS 5 μym, 250 × 4.5 mm column and a mobile phase mixture of 59:40:1 methanol:water:tetrahydrofuran separates the enprostil diastereomeric pairs and also resolves the enprostil diastereomers from potential interferences arising from placebo and drug degradation. The method performs well as evidenced by the usual statistical tests for: response linearity, recovery efficiency, precision, and lower quantitation limit. Also, the demonstrated dependence of chromatographic retention on mobile phase composition and system operating parameters defines acceptable operating condition ranges.     

Analysis of the aqueous phase of human cervical mucus by reversed-phase high-performance liquid chromatography and capillary isotachophoresis

The aqueous phase of human cervical mucus was analysed by reversed-phase high-performance liquid chromatography (HPLC) and capillary isotachophoresis (ITP). With HPLC, seventeen ultraviolet-absorbing and eight fluorescent components and with ITP five anionic and four cationic components could be determined. The sample pre-treatment consisted of a simple ultrafiltration. Ten samples from fertile women and eleven samples from infertile women were analysed. In six samples from the infertile group higher median concentrations of several components were found. This may be an indication of disturbances in the biochemical processes of the cervical mucus of woman with fertility problems.     

固相萃取-高效液相色谱法同时测定鸡粪中四环素类、喹诺酮类和磺胺类抗生素

建立了一种高效、低成本的固相萃取-高效液相色谱（SPE-HPLC）同时测定鸡粪中6种常见抗生素（2种四环素类、2种喹诺酮类和2种磺胺类）的分析方法。样品经EDTA-McIlvaine缓冲液和有机混合提取液（甲醇-乙腈-丙酮，2：2：1，v/v/v）提取，过HLB固相萃取柱净化，甲醇-二氯甲烷（7：3，v/v）洗脱，高效液相色谱-二极管阵列检测器测定，检测波长λ=270 nm，柱温32℃，流动相为乙腈-0.7%（v/v）磷酸水溶液。该方法在0.5~100 mg/L质量浓度范围内的标准曲线相关系数r²在0.9999~1之间，样品加标回收率在70.0%~116.3%之间，相对标准偏差为1.2%~16.6%。方法检出限为1.3~6.7 μg/kg，定量限为3.5~9.2 μg/kg。应用该方法对辽宁省抚顺市某养鸡场当天的鸡粪进行检测，诺氟沙星和恩诺沙星（喹诺酮类）含量为未检出~9.23 mg/kg和1.57~7.69 mg/kg，磺胺二甲嘧啶（磺胺类）含量为2.02~13.05 mg/kg，磺胺甲恶唑、土霉素和四环素未测出。     

Electrochemical oxidation of tetracycline on a boron doped diamond electrode within the stability potentials of water

Electrochemical oxidation of tetracycline on a boron doped diamond electrode within the stability potentials of water was studied in order to develop an approach for the purification of waste water containing medicinal agents. Cyclic voltammetry, HPLC, and high resolution mass spectrometry were used to establish that in the electrochemical oxidation process, tetracycline adds one oxygen atom to further form organic compounds with molecular weights higher than that of tetracycline. It was found that tetracycline in this region of potentials can be almost completely deactivated without its mineralization.     

A method for the low-level (ng g(-1)) determination of perfluorooctanoate in paper and textile by liquid chromatography with tandem mass spectrometry

The determination of perfluorooctanoate (PFO) in articles of commerce has become increasingly important to understand if treated products are a possible source of PFO. An LC-MS/MS method for the determination of PFO in paper and textile using a dual labeled 13C-PFOA internal standard was successfully developed and validated. Residues of PFO were determined using an isocratic, reversed-phase high-performance liquid chromatography (HPLC) method with an ammonium acetate/methanol buffer. Ions monitored were 413 (parent) and 369 (daughter) for PFO and 415 (parent) and 370 (daughter) for dual labeled 13C-PFOA internal standard. As a precaution against ubiquitous PFO that occasionally occurs in mobile phase or instrument components, two Hypercarb cartridges (4 mm) were placed before the HPLC injector. Any PFO that was captured by the cartridges was removed before each injection by flushing the system with 100% methanol prior to equilibration with the isocratic mobile phase. Overall recovery and standard deviation over a 3 day validation regimen for samples (n=54-55) fortified with PFOA at 5, 50, and 200 ng g(-1) were 114+/-4.9% for textile and 110+/-7.6% for paper. The results also established a limit of detection (LOD) of 1 ng g(-1) in textile and 2 ng g(-1) in paper based upon S/N of the 5.0 ng g(-1) fortification versus the untreated paper and textile.