The potential of pre-columns to improve detection properties in high-performance liquid chromatography

Summary The potential of pre-columns to increase the selectivity and sensitivity in high-performance liquid chromatography is demonstrated by some case studies. An on-line pre-column of 5 cm was used for the determination of fluvoxamine and clovoxamine in plasma. In this example, the principle of using different packing materials in precolumn and analytical column is shown to be promising for certain applications of pre-columns for clean-up and pre-concentration. An on-line pre-column containing a 1–2 mm layer of stationary phase was successfully applied for the determination of secoverine in plasma and serum. Direct injection of 200–1000 l of the sample is possible. This study illustrates the high selectivity obtained by the combined use of a pre-column and an extraction detector. Sep-Pak C₁₈ cartridges were used both for clean-up and pre-concentration of soil extracts in order to isolate radio-labelled pesticides. In this way, the pretreatment of such complex samples is very rapid and simple.     

Adjustment of a GC automatic liquid sampler for high-performance liquid chromatography

Summary An automatic liquid sampler for high-performance liquid chromatography is described.The sampler is based on a GC-injection system from Hewlett-Packard. The HPLC sampler has a unique cleaning system, excellent reproducibility and a plug-in compatibility for sample identification and computer control for Hewlett-Packard integrators and Laboratory Data Systems.     

Comparison of several curve resolution methods for drug impurity profiling using high-performance liquid chromatography with diode array detection

The performance of five curve resolution methods was compared systematically for the identification and quantification of impurities in drug impurity profiling. These methods are alternating least-squares (ALS) with either random or iterative key-set factor analysis (IKSFA) initialisation, iterative target transformation factor analysis (ITTFA), evolving factor analysis (EFA), and heuristic evolving latent projections (HELP). Real and simulated high-performance liquid chromatography diode array detection (HPLC-DAD) data were obtained for drug mixtures containing one main compound and two impurities. The elution order of the main compound and the impurities was varied. Furthermore, resolutions were varied from 0.56 to 3.36 and impurity levels from 30% down to 0.1%. For simulated data, ALS with IKSFA initialisation and HELP perform better than ITTFA and EFA, which perform better than ALS with random initialisation. ITTFA works better than EFA for almost completely separated data, while the opposite is true for moderately or strongly overlapping data. Only ALS with IKSFA initialisation and HELP were found to resolve the required 0.1% level for moderately overlapping data. For real data, comparison of the methods provides similar results. ITTFA performs clearly better than EFA. However, none of the curve resolution methods can identify or quantify impurities at the required 0.1% level. The results for real data are worse than for simulated data because of heteroscedasticity, nonlinearity, and the acquisition resolution of the A/D-converter.     

Determination of glutathione content in grape juice and wine by high-performance liquid chromatography with fluorescence detection

A modified preparation of sample was developed for the determination of glutathione content in grape juice and wine by high-performance liquid chromatography with fluorescence detection, using on-line pre-column derivatization. Ice-cold deoxygenated methanol was used to deactivate the oxidation enzymes in juices or wines and keep the glutathione stable. The optimum recovery of glutathione content in grape juice and wine was obtained when either the sample of grape juice or wine was mixed in ice-cold deoxygenated methanol in the ratio 10:90 (v:v) and further diluted in sodium acetate buffer in the ratio 1:1 (v:v). The optimized method was validated for linearity, limit of detection, limit of quantification, precision and uncertainty. According to the validation data the method is appropriate for the determination of glutathione content in grape juice and wine. Glutathione contents in grape juices made from White Muscat grapes and Sauvignon Blanc wines were analysed. The average glutathione content in 28 young Sauvignon Blanc wines was 12.5 mg L⁻¹.     

Determination of methoxsalen in dosage forms by high-performance liquid chromatography

Summary A new, rapid, sensitive, and specific method for the determination of methoxsalen in dosage forms using HPLC has been developed. methoxsalen is extracted in chloroform, evaporated on a water bath, and the residue is redissolved in ethanol. A standard solution of khellin (internal standard) in ethanol is added, and injected. A plot of peak height ratio (methoxsalen/internal standard) vs. concentration of methoxsalen gave a straight line (r=0.998). The column used was a stainless steel, 3.8 mm×30 cm, and the mobile phase was methanol: water (6040) at a flow rate of 2 cm³/min. Retention times for methoxsalen and khellin were 3.45 and 9.6 min, respectively. This method was found superior to the spectrophotometric assay in that no interference was encountered from structurally similar compounds or from coloring agents used in some commercial methoxsalen products.     

Second-order multivariate calibration procedures applied to high-performance liquid chromatography coupled to fast-scanning fluorescence detection for the determination of fluoroquinolones

Different second-order multivariate calibration algorithms, namely parallel factor analysis (PARAFAC), N-dimensional partial least-squares (N-PLS) and multivariate curve resolution-alternating least-squares (MCR-ALS) have been compared for the analysis of four fluoroquinolones in aqueous solutions, including some human urine samples (additional four fluoroquinolones were simultaneously determined by univariate calibration). Data were measured in a short time with a chromatographic system operating in the isocratic mode. The detection system consisted of a fast-scanning spectrofluorimeter, which allows one to obtain second-order data matrices containing the fluorescence intensity as a function of retention time and emission wavelength. The developed approach enabled us to determine eight analytes, some of them with overlapped profiles, without the necessity of applying an elution gradient, and thus significantly reducing both the experimental time and complexity. The study was employed for the discussion of the scopes of the applied second-order chemometric tools. The quality of the proposed technique coupled to each of the evaluated algorithms was assessed on the basis of the figures of merit for the determination of fluoroquinolones in the analyzed water and urine samples. Univariate calibration of four analytes led to limits of detection in the range 20–40 ng mL⁻¹ and root mean square errors for the validation samples in the range 30–60 ng mL⁻¹ (corresponding to relative prediction errors of 3–8%). The ranges for second-order multivariate calibration (using PARAFAC and N-PLS) of the remaining four analytes were: limit of detection, 2–8 ng mL⁻¹, root mean square errors, 3–50 ng mL⁻¹ and relative prediction errors, 1–5%.     

高效液相色谱法准确测定血清中的尿酸

建立了准确测定血清中尿酸含量的高效液相色谱方法,血清样品利用乙腈沉淀蛋白,过滤后直接进样测定。所采用的色谱柱为Inertsil ODS-SP(4.6mm i.d.×250 mm,5μm),柱温25℃,流动相体系为10 mmol/L乙酸铵(pH 4.5),流速为1.0 mL/min,紫外检测波长为280 nm。线性范围12.5~150μg/g,回收率为99.79%~100.5%。本文采用乙酸铵缓冲体系,对环境的污染小,同时也为建立液相色谱-质谱法测定尿酸奠定了良好的基础。     

A reliable high-performance liquid chromatography with ultraviolet detection for the determination of sulfonamides in honey

The sulfonamides are stable chemotherapeutics used against the bacterial disease affecting bees, known as American foulbrood (Bacillus larvae), so their residues could appear in the honey of treated bees. Their presence at a concentration above the limit value is a potential hazard to human health. Brazilian authorities have included in the National regulatory monitoring program, the control of the three most widely used sulfonamides in honey production, i.e., sulfathiazole, sulfamethazine and sulfadimethoxine. A method for the determination of residual sulfonamides in honey, using sulfapyridine as an internal standard has been developed, optimized and validated. Some changes were implemented on current available methodologies for the analysis of sulfonamides in honey in order to adopt such procedures to Brazilian honey samples. Sulfonamides were extracted from honey with dichloromethane after dissolution with 30% sodium chloride, and cleaned up with solid phase extraction on Florisil columns. The eluate was analyzed by high-performance liquid chromatography with ultraviolet detection. The limit of detection was determined at 3 μg kg⁻¹, 4 μg kg⁻¹ and 5 μg kg⁻¹ for sulfathiazole, sulfamethazine and sulfadimethoxine, respectively with average recoveries of 61.0% for sulfathiazole; 94.5% for sulfamethazine and 86.0% for sulfadimethoxine at the 100 μg kg⁻¹ level. As the final step of validation procedure, the analysts were submitted to a blind spiked sample prepared by the quality assurance officer which results were successfully obtained regarding recovery and deviations.     

Reversed-phase systems for the separation of pentapeptides by high-performance liquid chromatography

Summary Reversed-phase systems using octyl modified silica as such and as a support for dynamically coated ion-exchangers, were investigated for their ability to separate pentapeptides. Normal reversed-phase adsorption with C-8 bonded silica in combination with citrate bufferpropanol-1 mixtures were found useful for the separation of a number of pentapeptides. The separation of pentapeptides differing widely in retention can be speeded up by applying an organic modifier and/or sodium citrate gradient. A solvent generated cation-exchange system with sodium dodecylsulfate as surfactant showed a high selectivity for the pentapeptides under investigation and is better for analytical purposes than the normal reversed-phase adsorption systems investigated. With respect to the detection of pentapeptides with fluorescamine, the use of dry pyridine as a basic buffer and as diluent for the fluorescamine was also investigated. Compared to the commonly used diluent acetone, pyridine is better when using acidic eluents of moderate buffer strength. At pH>6 no significant differences in sensitivity between acetone and pyridine could be noticed.     

Determination of vitamin K homologues by high-performance liquid chromatography with on-line photoreactor and peroxyoxalate chemiluminescence detection

A sensitive and highly selective high-performance liquid chromatography (HPLC) method was developed for the determination of vitamin K homologues including phylloquinone (PK), menaquinone-4 (MK-4) and menaquinone-7 (MK-7) in human plasma using post-column peroxyoxalate chemiluminescence (PO-CL) detection following on-line ultraviolet (UV) irradiation. The method was based on ultraviolet irradiation (254 nm, 15 W) of vitamin K to produce hydrogen peroxide and a fluorescent product at the same time, which can be determined with PO-CL detection. The separation of vitamin K by HPLC was accomplished isocratically on an ODS column within 35 min. The method involves the use of 2-methyl-3-pentadecyl-1,4-naphthoquinone as an internal standard. The detection limits (signal-to-noise ratio = 3) were 32, 38 and 85 fmol for PK, MK-4 and MK-7, respectively. The recoveries of PK, MK-4 and MK-7 were greater than 82% and the inter- and intra-assay R.S.D. values were 1.9-5.4%. The sensitivity and selectivity of this method were sufficient for clinical and nutritional applications.     

The determination of atmospheric concentrations of the active ingredients of pesticide formulations by high-performance liquid chromatography

Summary Methods of sampling atmospheres contaminated by pesticides in factory and agricultural environments, and subsequent analysis by HPLC, are discussed. Air sampling is carried out using porous polymer or filter collection media, usually a 100 dm³ air volume is suitable. Detection limits with ultra-violet detection are in the range 0.1 to 10 g m^–3.Presented at the 14th International Symposium on Chromatography London, September, 1982     

Determination of macrolide antibiotics in porcine and bovine urine by high-performance liquid chromatography coupled to coulometric detection

A high-performance liquid chromatography method coupled to coulometric detection has been applied for the determination, in a single run, of up to eight macrolide antibiotics (erythromycin [ERY], tylosin [TYL], tilmicosin [TILM], spiramycin 2 [SPI 2], spiramycin 3 [SPI 3], josamycin [JOS], kitasamycin [KIT], and rosamicin [ROS]) in spiked porcine and bovine urine. Quantification was performed using matrix-matched calibration with roxithromycin (ROX) as the internal standard. The detection limits for each drug were below 3.5 ng injected (equivalent to an initial concentration below 0.07 mg L^–1) for porcine urine and below 5 ng injected (equivalent to an initial concentration below 0.10 mg L^–1) for bovine urine. Recoveries from urine samples spiked at three different concentrations within the linear range were not significantly dependent on concentration. The entire procedure provides average macrolide recoveries ranging from 69.7 to 96.6% for bovine urine and from 75.5 and 96.1% for porcine urine.     

高效液相色谱荧光测定及质谱鉴定分析血清中胆汁酸

利用新型荧光试剂1,2-苯并-3,4-二氢咔唑-9-乙基苯磺酸酯(BDEBS)作柱前衍生化试剂,在EclipseXDB-C8色谱柱上,采用梯度洗脱对10种胆汁酸(BAs)荧光衍生物进行了优化分离。95℃下在二甲基亚砜(DMSO)溶剂中以柠檬酸钾作催化剂,衍生反应35min后获得稳定的荧光产物,衍生反应完全。荧光激发和发射波长分别为λex=333nm,λem=390nm。采用大气压化学电离源(APCISource)正离子模式,实现了血清中胆汁酸的定性测定。分析物的定量测定采用荧光法进行。线性回归系数均在0.9996以上,检出限为22.36~44.57×10-15mol。     

A simple,corrosion-resistant flow cell for laser-induced photoacoustic spectroscopic detection of high-performance liquid chromatography effluents

Summary A simple, corrosion-resistant and non-destructive flow cell optimized for laser induced photoacoustic spectroscopic detection of HPLC effluents is described. The characteristics and analytical figures of merit of the flow cell are presented. Application of the flow cell to the detection of polynuclear aromatic hydrocarbons in HPLC effluent is demonstrated. Limits of detection were on the order of tenths of parts per million. Comparisons with the conventional UV absorbance detector and among various laser powers are discussed.     

Synthesis of silica-based benzeneboronic acid affinity materials and application as pre-column in coupled-column high-performance liquid chromatography

Three silica-based benzeneboronic acid affinity materials were synthesized by using an m-aminobenzeneboronic acid as the ligand and using three different spacer arms. Under high-pressure, three affinity pre-columns were packed with these materials and the retention of every affinity pre-column with 11 urinary nucleosides was studied. With different spacer arms of boronic acid-substituted silica materials, the absorption to vicinal alcohols (cis-diols) and stability under acidic elution conditions are of great difference. Coupled-column liquid chromatographic methods for the direct analysis of urinary nucleosides were respectively established. As a result, two of three affinity pre-columns showed good chromatographic property in the on-line analysis of urinary nucleosides. The coupled-column system including pre-column I is the best with excellent linearity (R² > 0.995), good recoveries (85.6–96.9%) and reproducibility (R.S.D.: 1.01–4.02%). The pre-column I could at least endure 150 repetitive injections of a 100 μL urinary sample.     

Analysis of triazine in water samples by solid-phase microextraction coupled with high-performance liquid chromatography

Solid-phase microextraction (SPME) coupled with high-performance liquid chromatography (HPLC) for the determination of triazine is described. Carbowax/templated resin (CW/TPR, 50 μm), polydimethylsiloxane/divinylbenzene (PDMS/DVB, 60 μm), polydimethylsiloxane (PDMS, 100 μm), and polyacrylate (PA, 85 μm) fibers were evaluated for extraction of the triazines. CW/TPR and PDMS/DVB fibers were selected for further study. Several parameters of the extraction and desorption procedure were studied and optimized (such as types of fibers, desorption mode, desorption time, compositions of solvent for desorption, soaking periods and the flow rate during desorption period, extraction time, temperature, pH, and ionic strength of samples). Both CW/TPR and PDMS/DVB fibers are acceptable; a simple calibration-curve method based on simple aqueous standards can be used. The linearity of this method for analyzing standard solution has been investigated over the range 5-1000 ng mL⁻¹ for both PDMS/DVB and CW/TPR fibers. All the correlation coefficients in the range 5-1000 ng mL⁻¹ were better than 0.995 except Simazine and Atratone by CW/TPR fiber. The R.S.D.s range from 4.4% to 8.8 % (PDMS/DVB fiber) and from 2.4% to 7.2% (CW/TPR fiber). Method-detection limits (MDL) are in the range 1.2-2.6 and 2.8-3.4 ng mL⁻¹ for the two fibers. These methods were applied to the determination of trazines in environmental water samples (lake water).     

Fingerprint analysis of Dioscorea nipponica by high-performance liquid chromatography with evaporative light scattering detection

High-performance liquid chromatographic method (HPLC) with evaporative light scattering detection (ELSD) coupled with microwave-assisted extraction (MAE) as an efficient sample preparation technique has been developed for fingerprint analysis of Dioscorea nipponica. The samples were separated with an Agilent C8 column using water (A) and acetonitrile (B) under gradient conditions (0-10 min, linear gradient 20-40% B; 10-12 min, linear gradient 40-42% B; 12-25 min, isocratic 42% B) as the mobile phase at a flow rate of 1 mL min⁻¹ within 22 min. The ELSD conditions were optimized at nebulizer-gas flow rate 2.7 L min⁻¹ and drift tube temperature 90 °C. Precision experiments showed relative standard deviation (R.S.D.) of peak area and retention time were better than 2.5%; inter-day and intra-day variabilities showed that R.S.D. was ranged from 0.78% to 4.74%. Limit of detection was less than 50 μg mL⁻¹ and limit of quantification was less than 80 μg mL⁻¹. Accuracy validation showed that average recovery was between 97.39% and 104.07%. The method was validated to achieve the satisfactory precision and recovery. Relative retention time and relative peak area were used to identify the common peaks for fingerprint analysis. There are nine common peaks in the fingerprint. The quality of seven batches of D. nipponica samples was evaluated to be qualified or unqualified by the parameters “difference” and “total difference” of common peaks. Furthermore, the contents of important medicinal compounds (dioscin, prodioscin and gracillin) in different batches of D. nipponica samples were determined simultaneously using the developed HPLC-ELSD method. The results indicated variation of the herb quality which might be related to different producing area, growing condition, climate, harvest time, drug processing and so on. The developed analytical procedure was proved to be a reliable and rapid method for the quality control of D. nipponica.     

Determination of risedronate in rat plasma samples by ion-pair high-performance liquid chromatography with UV detector

In the present work, an analytical method for determination of risedronate, a member of bisphosphonates, is described for the routine analysis in rat plasma. Sample pre-treatment involves protein precipitation, co-precipitation with calcium at alkaline pH, hydrolysis of possible derivatives of pyrophosphate and reprecipitation. A good separation was obtained by using a reversed-phase column (Hypersil ODS-2 C₁₈, 4.6 mm × 250 mm, 5 μm). The mobile phase was an aqueous solution of buffer (contained 1.5 mM EDTA-2Na, 1 mM sodium etidronate, 11 mM sodium phosphate and 5 mM tetrabutylammonium bromide as ion-pair reagent) - methanol (88:12, v/v) adjusted to pH 6.75 using 1 M NaOH. The flow rate was 1 ml min⁻¹. UV detection (λ = 262 nm) was used to quantitate risedronate in the concentration range of 10-500 ng ml⁻¹. The limit of detection and quantitation for risedronate were 7 and 10 ng ml⁻¹, respectively. The method was applied successfully to plasma samples from Wistar rats undergoing oral administration of risedronate mini-pills. Precision, extraction recoveries, as well as accuracy results, were satisfactory and no interference was found at the retention time of risedronate. Hence, the method is suitable for monitoring risedronate in rat plasma.     

Analysis of meloxicam by high-performance liquid chromatography with cloud-point extraction

A simple cloud-point extraction method for the determination of meloxicam in human serum was developed. Meloxicam was extracted from serum sample after adding 1 mL of 3% (v/v) Triton X-114 aqueous solution in the presence of 1M HCl and 60 mg NaCl. The meloxicam, present in the surfactant-rich phase, was enriched again with acetonitrile. Tenoxicam was used as the external standard. The separation was achieved on a C18 analytical column with a mobile phase consisting of aqueous acetic acid (1%, v/v) and acetonitrile (54:46, v/v). UV detection was performed at 360 nm. The response was linear over the range 45–2000 ng mL⁻¹ in human serum, and intra- and interday precisions of less than 15.0% were obtained. The relative error was within ±3.0%. The recoveries of meloxicam were larger than 92.0%. The method was compared with liquid–liquid extraction. The results showed that the new method has a considerable LOQ and higher recoveries but poorer precision than liquid–liquid extraction, which exhibited poor recoveries of less than 86.0%, precisions of less than 5.0% and relative errors of less than 7.0%. The method was used for the determination of meloxicam in healthy human volunteers.     

Comparison of microemulsion electrokinetic chromatography with high-performance liquid chromatography for fingerprint analysis of resina draconis

Microemulsion electrokinetic chromatography (MEEKC) has been developed for fingerprint analysis of resina draconis, a substitute for sanguis draconis in the Chinese market. The microemulsion as the running buffer was made up of 3.3% (w/v) sodium dodecyl sulfate (SDS), 6.6% (w/v) n-butanol, 0.8% (w/v) n-octane, and 10 mmol/L sodium tetraborate buffer (pH 9.2), which was also used as the solvent for ultrasonic extraction of both water- and fat-soluble compounds in the traditional Chinese medicine samples. Four batches of resina draconis obtained from different pharmaceutical factories located in different geographic regions were used to establish the electrophoretic fingerprint. MEEKC was performed using a Beckman PACE/MDQ system equipped with a diode-array detector and with monitoring at 280 nm. The fingerprint of resina draconis comprised 27 common peaks within 100 min. The relative standard deviations of the relative migration time of these common peaks were less than 2.1%. Through repetitive injection of the sample solution six times in 24 h, all relative standard deviations of the migration time and peak area of loureirin A and loureirin B were less than 2.5 and 3.8%, which demonstrated that the method had good stability and reproducibility. The relative peak areas of these common peaks in the electropherograms of four batches of resina draconis were processed with two mathematical methods, the correlation coefficient and the interangle cosine, to valuate the similarity. The values of the similarity degree of all samples were more than 0.91, which showed resina draconis samples from different origins were consistent. On the other hand, high-performance liquid chromatography (HPLC) coupled with photodiode-array detection was also applied to establish the fingerprint of resina draconis. The samples were separated with a LiChrospher C₁₈ column using acetonitrile (solvent A) and water containing 0.1% H₃PO₄ (solvent B) as the mobile phase in linear gradient elution mode at a flow rate of 0.6 mL/min and detection was at 280 nm. There were only 20 common peaks in the HPLC fingerprint, and the values of the similarity degree of all samples were also more than 0.91. Though the similarity results of fingerprint analysis seemed to be the same, MEEKC resulted in more common peaks and higher separation efficiency for a variety of polarities of the components than HPLC. So, MEEKC was more suitable for development of the fingerprint of resina draconis.