Automatic High-performance Liquid Chromatography Research Articles

Impact of insulin adsorption in various containers during hyperkalaemia treatment

BackgroundInsulin–glucose therapy in hyperkalaemia treatment has a narrow therapeutic index for a safe and efficient use. We assess the variability of the effective delivered insulin under conditions used in the setting of hyperkalaemia treatment.MethodsA range of simulated insulin infusions was studied using different containers (bag or syringes) according to the different hyperkalaemia treatment procedures of our institution. Insulin concentration was assayed using a chromatographic method on an automatic high-performance liquid chromatography. We calculated the effective delivered insulin and compared the time average of percentage delivered insulin (TAdi) between all the procedures.ResultsThe TAdi was significantly decreased to 63.3% of the expected insulin delivery in the polyurethane (PE) bag compared with allover container. The procedure duration and the insulin concentration influenced the variability of the insulin delivery in the PE and glass bag. The polyvinyl chloride bag had the highest TAdi at 93.8%, without significant variation during the time. TAdi reaches ∼90% of the expected insulin with all the syringe procedure without variation according to the solute used to dilute insulin.ConclusionsClinically significant variations in intravenous insulin delivery occur in the setting of hyperkalaemia treatment according to the container. The use of propylene syringe limits the insulin delivery variation. In the future, clinical studies on hyperkalaemia treatment by insulin–glucose therapy should detail the procedure precisely.

Update in Laboratory Diagnosis of Thalassemia.

Alpha- and β-thalassemias and abnormal hemoglobin (Hb) are common in tropical countries. These abnormal globin genes in different combinations lead to many thalassemic diseases including three severe thalassemia diseases, i.e., homozygous β-thalassemia, β-thalassemia/Hb E, and Hb Bart’s hydrops fetalis. Laboratory diagnosis of thalassemia requires a number of tests including red blood cell indices and Hb and DNA analyses. Thalassemic red blood cell analysis with an automated hematology analyzer is a primary screening for thalassemia since microcytosis and decreased Hb content of red blood cells are hallmarks of all thalassemic red blood cells. However, these two red blood cell indices cannot discriminate between thalassemia trait and iron deficiency or between α- and β-thalassemic conditions. Today, Hb analysis may be carried out by either automatic high-performance liquid chromatography (HPLC) or capillary zone electrophoresis (CE) system. These two systems give both qualitative and quantitative analysis of Hb components and help to do thalassemia prenatal and postnatal diagnoses within a short period. Both systems have a good correlation, but the interpretation under the CE system should be done with caution because Hb A2 is clearly separated from Hb E. In case of α-thalassemia gene interaction, it can affect the amount of Hb A2/E. Thalassemia genotypes can be characterized by the intensities between alpha-/beta-globin chains or alpha-/beta-mRNA ratios. However, those are presumptive diagnoses. Only DNA analysis can be made for specific thalassemia mutation diagnosis. Various molecular techniques have been used for point mutation detection in β-thalassemia and large-deletion detection in α-thalassemia. All of these techniques have some advantages and disadvantages. Recently, screening for both α- and β-thalassemia genes by next-generation sequencing (NGS) has been introduced. This technique gives an accurate diagnosis of thalassemia that may be misdiagnosed by other conventional techniques. The major limitation for using NGS in the screening of thalassemia is its cost which is still expensive. All service labs highly recommend to select the technique(s) they are most familiar and most economic one for their routine use.

The Development of an Automated Clean-up for Fat Extracts in the Routine Analysis of Organochlorine Compounds in Fish Meat

The present work describes the development of a new, automatic High Performance Liquid Chromatography (HPLC) Clean-up step, in the methodology of sample preparation and multi-residue determination of organochlorine compounds (OCs) in fish meat. 24 OCs were taken into study. In addition 7 Polychlorinated Biphenyls (PCBs), 7 chlorobenzene compounds and one 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) were investigated. The HPLC conditions were established in accordance with the validated traditional Clean-up step of the laboratory. The technique was applied on a dilution of analytes of interest in order to establish the period of time in which the compounds are eluted. Another set of experiments involved fish oil, in order to identify and separate the fat fraction from the analytes. To confirm the findings of the experiments mentioned above, extracts of fish samples obtained after Accelerated Solvent Extraction (ASE) were examined. The samples were spiked with the analytes of interest before HPLC clean-up step and quantified through Gas Chromatography coupled with tandem Mass Spectrometry (GC-MS/MS). A HPLC clean-up technique lasting 38 minutes/sample was developed. The method is not suitable for OCs such as Endosulfansulfat and Endrine Ketone due to the very low recovery results.Â

A novel liquid chromatography method using diode-array detector for the determination of oleuropein in dietary supplements

A simple and fast chromatographic method using ultraviolet diode-array detector (UV-DAD) was developed for the automatic high performance liquid chromatography (HPLC) determination of the title of oleuropein in a new dietary supplements in form of effervescent granules. The chromatographic separations were performed on a C18 core-shell column with detection at λ=232nm. The mobile phase consisted of deionized water with 0.1% TFA and acetonitrile under gradient conditions at a flow-rate of 0.8mL/min. Oleuropein and oleuroside present in the raw material were characterized by high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI–MS/MS). The validation of the analytical procedure has been performed determining the following parameters: specificity, linearity, repeatability, reproducibility, accuracy, limit of quantification (LOQ), stability of the standard and sample solutions. Linear response was observed in fortified placebo solutions (determination coefficient: 0.9998). Intra-day precision (relative standard deviation, RSD) was ≤5.0% for peak area and for retention times (tR) without significant differences between intra- and inter-day data. The limits of quantitation (LOQ) was about 5μg/mL and 9pmol/inject. Oleuropein recovery studies gave good results (99.9%) with a R.S.D. of 0.5%. The speed of analysis and the stability of the solutions with a fluctuation Δ (%) ≤2.0 at room temperature means an undoubted advantage of the method allowing the simultaneous preparation of many samples and consecutive chromatographic analyses by using an autosampler. The developed method is suitable for the quality control of oleuropein in raw material and industrial products. The method can be applied in any analytical laboratory not requiring a sophisticated instrumentation.

Selective and Sensitive Determination of Mercury(II) Ions in River Water by an Automatic HPLC System Combined with On-Line Column Enrichment as 2-Mercaptopyrimidine Chelate

A selective and sensitive determination method for mercury (Hg) ions in river water by reversed phase high performance liquid chromatography (HPLC) has been developed. Two mL of water sample containing Hg(II) ions was put into a 4 mL sample tube. To the sample tube, 500 µL of 2 mol/L (M) acetate buffer solution (pH 4.5), and 50 µL of 0.01 M 2-mercaptopyrimidine (MP) were added to form a neutral Hg chelate. The Hg chelate subsequently became preconcentrated in a reversed phase mini column. By switching the valve, the Hg chelate was separated and detected at 255 nm. These processes occur automatically, except for the addition of water samples, buffer, and MP solutions. The correlation coefficients of the calibration curves obtained with 2 mL of mercury standard were more than 0.999 over the range of 20 ng/mL (ppb) to 10 µg/mL (ppm). The detection limit of Hg ions in 2 mL solution was 1.6 ppb, which corresponded to 3 times the standard deviation (N = 10) of the blank peak area. Effects of foreign ions on the determination of 0.2 ppm Hg were investigated with 57 metal ions. Almost none of the ions interfered except for Au(III), Pd(II), Ag(I), and Co(II) ions. The recoveries with spiked river water samples for 5, 0.5, and 0.05 Hg ion (N = 7) were 99.7 ± 0.4%, 100.3 ± 0.3%, and 100.7 ± 0.7%, respectively.

Selective and Sensitive Determination of Mercury(II) Ions in River and Sea Water by an Automatic HPLC System Combined with On-line Column Enrichment

A selective and sensitive determination method for mercury (Hg) ions in river and sea water by reversed‐phase high performance liquid chromatography (HPLC) has been developed. A one milliliter water sample containing Hg(II) ions was put into a 4 mL sample tube. To the sample tube, 2 mL of 0.5 mol/L (M) tetrabutylammonium bromide (TBA‐Br) solution was added to form a neutral Hg compound. The Hg compound subsequently became pre‐concentrated in a mini‐column packed with 5 µm ODS. By switching the valve, the Hg compound was separated in an ODS analytical column, and detected with a photometric detector (255 nm). These processes occur automatically, except for the addition of water samples and 0.5 M TBA‐Br. The correlation coefficient of the calibration curves (metal concentration vs. peak area) obtained with 1 mL of mercury standard was more than 0.9995 over the range of 1 ng/mL (ppb) to 10 µg/mL (ppm). The detection limit of Hg ions in 1 mL solution was 0.5 ppb, which corresponded to 3 times the standard deviation (N=8) of the blank peak area. Effects of foreign ions on the method were investigated with 0.1‐ppm Hg standard and 55 metal ions. Forty two metal ions of 200 ppm did not interfere with the determination of Hg(II) ions. Tolerance limits of Pd(II) and Ag(I) were 5 and 10 ppm, respectively. Recovery tests were carried out by the presented HPLC method with spiked river and sea water samples. The recoveries with a spiked river water sample for 400‐, 40‐, and 4‐ppb Hg ion (N=5) were 100.1±0.2%, 100.1±1.3%, and 97.9±2.3%, respectively. Those with a sea water sample for 400‐, 40‐, and 4‐ppb Hg ion (N=5) were 100.9±1.0%, 99.9±1.8%, and 99.1±9.4%, respectively.

Rapid analysis of 6-methoxymellein in carrots by boiling water extraction, solid phase extraction and HPLC

By modification and combination of existing methods we have developed a rapid method for quantification of 6-methoxymellein in carrot roots by means of boiling water and solid phase extraction, followed by high performance liquid chromatography (HPLC). The performance of the method was found to be acceptable, when tested by three consecutive boiling water extractions from carrot samples with different levels of 6-methoxymellein. The recovery was 96–99% after one extraction. With this method it was possible for one person to extract 20 samples during a working day and further quantify the amount of 6-methoxymellein by automatic HPLC the following night.

Highly sensitive automatic analysis of polycyclic aromatic hydrocarbons in indoor and outdoor air

A method for the quantitation of polycyclic aromatic hydrocarbons (PAHs) in indoor and outdoor air by high-performance liquid chromatography (HPLC) with a spectrofluorometric detection and programmed excitation and emission wavelength pairs is proposed. The mobile phase is a linear gradient of methanol-water. The relative standard deviations ( n = 5) are in the range 0.38–1.7% at concentration levels of 0.69–11.40 ng ml −1. The determination limits ( S N = 10 ) are 0.5–15.9 pg. The proposed method was successfully applied to quantitate 12 PAHs in gas phase and particulates in indoor and outdoor air. The recoveries of PAHs from gas phase and particulates were 95.7–117.5 and 94.8–112.4%, respectively. This highly sensitive automatic HPLC analysis for PAHs both in gas phase and particulates can be applied to indoor and outdoor survey.

Estimation of urinary unconjugated androstenedione, dehydroepiandrosterone, testosterone, cortisol, aldosterone and 18-hydroxycorticosterone as a potential tool for assessing adrenal status.

A method is described for the simultaneous assay of non-conjugated androstenedione, dehydroepiandrosterone, testosterone, cortisol, aldosterone and 18-hydroxycorticosterone in urine. The method involves solid-phase extraction, automatic high performance liquid chromatography and subsequent radioimmunological quantitation of the individual steroids. Excretion rates of these urinary free steroids were determined in normal males and females. There were no significant sex differences in excretion rates, although both urinary free testosterone and dehydroepiandrosterone were distinctly lower in females than in males. Representative measurements of the excretion rates of patients with Cushing's disease, Addison's disease, ectopic corticotropin syndrome and hirsutism were made. The present method has been shown to be well suited for routine purposes. Its final diagnostic significance for monitoring alterations in glucocorticoid, mineralocorticoid and androgenic activity of the adrenal cortex has yet to be explored.

Specific estimation of fifteen unconjugated, non-metabolized steroid hormones in human urine

A method for the specific determination of 15 free, non-metabolized steroid hormones in human urine is described. The steroids progesterone (P), androstenedione (AD), pregnenolone (PL), 5α-dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA), testosterone (T), 11-deoxycorticosterone (DOC), 17-OH-progesterone (17-OHP), 17-OH-pregnenolone (17-PL), 11-deoxycortisol (S), 18-OH-11-deoxycorticosterone (18-OH-DOC), corticosterone (B), aldosterone (Aldo), cortisol (F), and 18-OH-corticosterone (18-OH-B) were extracted from 2 ml urine samples by a solid-phase technique, subjected to automatic high performance liquid chromatography (HPLC) and finally quantitated by radioimmunoassay (RIA). The combination of HPLC and RIA provides a high specificity of steroid estimates. The automatic mode of HPLC renders the method quite suitable for series analyses in research or for routine purposes. Precision and accuracy are satisfactory, and comparable with the level commonly achieved in RIA techniques. The mean values (nmol/24 h) of reference ranges established from 32 normal males were as follows: P: 0.36; AD: 9.76; PL: 0.90; DHT: 0.61; DHEA: 6.76; T: 1.43; DOC: 0.35; 17-OHP: 1.03; 17-PL: 0.20; S: 0.24; 18-OH-DOC: 2.11; B: 1.49; Aldo: 0.46; F: 68.3; 18-OH-B: 5.41. This highly practicable method may be particularly useful for the further investigation of the physiological or diagnostic significance of the non-conjugated, non-metabolized fraction of steroid hormones in urine.

Complete computer automation of preparative liquid chromatography through intelligent fraction collection, with unlimited injection volume and repetitive collection of separated solute peaks

Most of the automatic high-performance liquid chromatography (HPLC) instruments, although comprising sophisticated injection and data systems, do not offer the possibility of achieving automatic preparative scale separations. To obtain the required capacity for unattended collection of the compounds separated, a programmable HPLC system controller (Waters 720) was interfaced between the detection system and a fraction collector. The computer program AUTOPREP, using BASIC language, was written to upgrade the standard 720 program, by automatically controlling the injection of large sample volumes and the collection of the peaks of solutes separated. Sample mixtures of unlimited volume could be injected through the pump head or, alternatively, small samples could be injected by the WISP autosampler, a process that has many advantages over the usual intensity threshold peak detection. A combination of contiuously adjusted peak slope thresholds and time windows provided logic decisions during teh chromatographic runs, which permitted to achieved various collection patterns. Finally, the model collection pattern generated by the program could be used for repetitive injections and collections. The practicability of the system is demonstrated with the automated isolation of compounds from sterol and phospholipid mixtures.

Estimation of free urinary aldosterone and 18-hydroxycorticosterone by a combination of automatic high-performance liquid chromatography and radio-immunoassay

Estimation of free urinary aldosterone and 18-hydroxycorticosterone by a combination of automatic high-performance liquid chromatography and radio-immunoassay

Quantitation of pregnenolone and 17-hydroxypregnenolone in human serum by automatic high-performance liquid chromatography and subsequent radioimmunoassay

A method for the determination of pregnenolone and 17-hydroxypregnenolone in human serum is described which uses high-performance liquid chromatography as a prepurification step followed by radioimmunological quantitation. As to specificity and practicability, the present technique is superior to previously reported methods. Chromatographic assessment of unspecific pregnenolone and 17-hydroxypregnenolone immunoreactivities arising in the ether extracts of normal serum samples clearly emphasizes the necessity of efficient chromatographic isolation of the steroids prior to immunoassay, if specific estimation is to be made. Normal values and physiological changes of serum pregnenolone and 17-hydroxypregnenolone accord well with the data already published in literature.

Assessment of eleven adrenal steroids from a single serum sample by combination of automatic high-performance liquid chromatography and radioimmunoassay (HPLC-RIA)

A radioimmunological method for the assessment of eleven adrenal steroids from one serum sample is presented. The method includes an extraction, purification and radioimmunological quantitation step. Automatic high-performance liquid chromatography (HPLC) is used as a Chromatographie techniquc for the purification step. The localization of steroid fractions during a Chromatographic run is triggered by the retention times established by chromatography of U.V.-detectable amounts of steroids prior to each batch. The main advantages of this automatic HPLC technique are (1) the high potency of resolution, (2) the high degree of reproducibility of retention times, (3) the gradient elution technique allowing “one run”-chromatography of compounds covering a great range of polarity, and (4) the automatic feature eliminating almost all manual operations. Specificity of the antisera raised against the steroids progesterone, pregnenolone, deoxycorticosterone, 17-OH-progesterone, 17-OH-pregnenolone, deoxycortisol, 18-OH-deoxycorticosterone, corticosterone, aldosterone, cortisol and 18-OH-corticoster-one were studied by cross-reactivity to known steroids and by monitoring the Chromatographic profiles of immunoreactivities arising in a normal serum sample. Base levels of serum steroids in normal man as well as their changes after adrenal stimulation and suppression were in accordance with the data reported in literature.