Ion-exchange HPLC Method Research Articles

Validated ion exchange HPLC method for the quantification of levothyroxine – a narrow therapeutic index drug – used for the treatment of hypothyroidism

Drugs with narrow therapeutic index (NTI-drugs) have been defined by the FDA as drugs with small differences between therapeutic and toxic doses that might lead to serious therapeutic failures or life-threatening adverse drug reactions. Levothyroxine sodium pentahydrate (LT4), a synthetic T4 hormone used for the treatment of hypothyroidism (a condition where there is a hormonal imbalance in the thyroid gland that is responsible for the regulation of several physiological, metabolic, cardiovascular, and neurological processes). LT4 is designated by the FDA as a narrow therapeutic index drug and is available in the market in the form of very low dose pharmaceutical formulations ranging from 25 mcg to 150 mcg. This requires that the pharmaceutical dosage form should contain the exact labeled amount of the active ingredient, LT4, such that safety and efficacy are maintained. Therefore, it is necessary to develop an a precise, accurate and sensitive analytical method for LT4 quantification considering the treatment doses being in micrograms. In the present work, an ion exchange HPLC method has been developed and validated for the determination of LT4 as per ICH guidelines. The developed method was found to be simple, sepcific, precise and accurate. The low LOD and LOQ values allowed the quantitfication of the active ingredient in different pharmaceutical products qualifying the method to be applied in quality control assays.

Separation of truncated basic fibroblast growth factor from the full-length protein by hydrophobic interaction chromatography

Basic fibroblast growth factor (FGF-2) is a labile protein which degrades over time or forms aggregates. A purification process combining a cation ion exchange chromatography (CIEX) capture step with a polishing step by hydrophobic interaction chromatography (HIC) efficiently depleted process-related impurities such as host cell proteins (HCPs), dsDNA and endotoxins from the clarified E. coli homogenate containing the overexpressed FGF-2. Fresh homogenates did not contain verifiable amounts of process-related impurities such as degraded product variants. However, the storage of the capture eluates for longer than two weeks at 4 °C led to product degradation up to 49%. Mass spectrometry showed that the truncated FGF-2 was devoid of the nine N-terminal amino acids. Removal of such product-related impurities is included in state of the art purifications because these byproducts bear the risk of having different biological activities. Therefore, the established purification process has been evaluated for its potential to deplete such truncated FGF-2 variants. The HIC polishing step using Toyopearl® Hexyl-650C with a linear salt gradient over 4 column volumes from 1.65 M trisodium citrate to 0 M separated this variant from the full-length FGF-2. The truncated form eluted before the full length product due to the loss of 9 hydrophobic amino acids. The developed ion exchange HPLC method enabled us to quantify the truncated variant and full-length FGF-2. A purity of > 98% full-length FGF-2 with a step yield of up 86% is achieved. As long as the degree of truncation does not exceed 25% of the stored eluates, the purification process delivers the product in the desired quality of > 98% full-length FGF-2, HCP content below 10 ppm/dose, dsDNA content below 10 ng/mL/dose and endotoxin level below 400 EU/dose. This purification sequence allowed the production of FGF-2 which meets distinctive quality standards defined by regulatory bodies with respect to the depletion of both - process- and product-related impurities.

High hydrostatic pressure processing enhances pectin solubilisation on apple by-product improving techno-functional properties

Apple by-product is an extensive and inexpensive residue rich in dietary fibre, mainly insoluble fraction. Regarding soluble fibre, pectins are the predominant component responsible for specific physicochemical characteristics which are intimately related to its functional properties. To valorise apple by-product enhancing the solubility of pectins, high hydrostatic pressure (HHP) processing at 200–600 MPa during 15 and 30 min under controlled temperature was performed. Release of soluble carbohydrates was monitored using ion-exchange HPLC method with refractive index detection and dietary fibre composition was studied. A value of 200 MPa during 15 min was enough to enhance the solubilisation of cell wall components, mainly pectins, increasing significantly (p < 0.05) about threefold high/medium molecular weight soluble carbohydrates and maintaining total phenolic content. HHP preserved brightness, yellowish hue and slightly decreased pH. Furthermore, this valorisation procedure upgraded emulsion activity and stability improving the technological aptitude and enhanced swelling, oil-holding and cation-exchange capacities suggesting a potential lipid-lowering effect.

Évaluation de la concordance des valeurs d'hémoglobine glyquée entre les automates de biochimie D-10® (Biorad) et Minicap Flex piercing® (Sebia)

Glycated hemoglobin (HbAlc) is a key marker in the management of diabetic patients, particularly for the monitoring of glycemic control. Today, various automated glycated hemoglobin assay systems are available on the market. Thus, in order to meet one of the requirements of the ISO 15189 standard, a study was carried out in order to evaluate the agreement between the results obtained by two biochemical machines, in particular the D-10® (Biorad) system based on an ion exchange HPLC method and the Minicap Flex Piercing® (Sebia) that uses the principle of capillary electrophoresis. In total, thirty samples covering the measurement interval defined by the suppliers were tested and for each sample the assays were performed in parallel on the two automata. We found a good correlation between the two systems with a coefficient R = 0.995 (p <0.0001). The difference diagram according to Bland and Altman shows a distribution of differences for both normal HbAlc values and high values (> 86 mmol/mol or 10%). Ninety-five percent of the differences are between - 0.93 and + 11.33 mmol/mol (or - 0.08% and + 1.01%). This large range of differences does not allow to affirm a transferability between the two systems and require the follow-up of patients with the same technique.

DIFFERENCE IN GLICATED HEMOGLOBIN LEVEL BETWEEN BORONATE AFFINITY METHOD AND ION EXCHANGE-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY METHOD IN TYPE 2 DIABETES MELLITUS

Background: Glycated Hb (HbA1c) test is needed to control glycemic in high prevalence type 2 diabetes mellitus (DM) patients. Hemoglobin fraction separated and chemical reaction are two main concepts in HbA1c test. Ion exchange-high performance liquid chromatography (HPLC) and boronate affinity use first concept. Ion exchange-HPLC is reference method in most of clinical laboratorium. Point of care testing (POCT) with boronate affinity method that has been standardized by international institution is available. The aim of this study was to compare boronate affinity POCT method and ion exchange-HPLC method.Method: This cross sectional study was conducted to 22 type 2 DM patients those fullfill inclusion and exclusion criteria in January 2017 to February 2018. Level of HbA1c was assayed with boronate affinity POCT and ion exchange-HPLC method. T-test was used to analyse data and no significance difference if p&gt;0.005Results: Subjects of this study are women (59.1%) more than men (40.9%) with age mean 59.23 years old (8.1). Uncontroled type 2 DM (77.3%) more than controled type 2 DM (22.7%).Mean of HbA1 level was 8.0% (1.7) in boronate affinity POCT and 8.3% (1.8) in ion exchange-HPLC. T-test showed no significance difference between those two HbA1C assay methods (p&gt;0.005).Conclusion: There was no difference HbA1c level between boronate affinity POCT method and ion exchange-HPLC method.

Hubungan resting metabolic rate (RMR) dan komposisi tubuh dengan kadar HbA1c pada obesitas

Background: Obesity occurs due to an imbalance intake and energy expenditure. The cause of obesity is low energy expenditure whereas the resting metabolic rate (RMR) is the largest component of energy expenditure of the body. The factors that influence RMR are body composition, age, sex and hormonal status. Body composition measured as fat mass and free fat mass (FFM). Body fat increase in obesity can cause insulin resistance which will increase blood glucose levels. This study aims to determine the correlation between RMR and body composition (fat mass and FFM) with HbA1c levels increase as a parameter of diabetes mellitus in obesity.Methods: Cross sectional study design to 38 subjects young adults obese. Resting metabolic rate and body composition (fat mass and FFM) measured with Omron Karada scan HBF 375. HbA1c measured with ion-exchange HPLC method. Statistical analysis using the Spearman’s correlation test (p&lt;0.05)Result: HbA1c mean of 5.4% is still a normal value of HbA1c. There were no correlation between RMR and HbA1c (p = 0.768), fat mass with HbA1c (p = 0.102) and FFM with HbA1c (p = 0.843).Conclusion: RMR and body composition (fat mass and FFM) are not associated with HbA1c levels in obese young adults.

Path Analysis of the Effect of Waist-Pelvic Circumference, Body Mass Index, and Abdominal Circumference on the Occurrence of Prediabetes

Background: Diabetes mellitus is an important health problem in the world. Pre-diabetes is a state of blood sugar levels above normal but below the criteria for diabetes. American Diabetes Association (ADA) uses criteria for hemoglobin A1C (HbA1C) levels of 5.7% to 6.4% to define pre-diabetes. The prevalence of pre-diabetes was the highest in overweight individuals. In many studies, body fat levels were assessed by indicators of waist-pelvic circumference, abdominal circumference, and BMI. Among the three, it is still a debate which is more influential on the condition of pre-diabetes. The purpose of this study was to determine the effects of waist-pelvic circumference, BMI, and abdominal circumference in pre-diabetes. Subjects and Method: A cross-sectional study was conducted at Prodia Clinic, Surakarta, Central Java, from January to March 2019. A sample of 200 study subjects was selected by fixed disease sampling. The dependent variable was pre-diabetes. The independent variables were the waist-pelvic circumference, BMI, and abdominal circumference. The data on HbA1C was measured by NGSP standardized ion-exchange HPLC method. The data were analyzed by path analysis. Results: Abdominal circumference >90 cm in men and >80 cm in women (b= 0.87; 95% CI= 0.23 to 1.51; p= 0.008) and age ≥45 years old (b = 1.70; 95% CI = 0.93 to 2.46; p 90 cm in men and >80 cm in women and age ≥45 years old are directly increased pre-diabetes. Pre-diabetes is indirectly affected by waist–pelvic circum­ference, gender, and obesity. Keywords: prediabetes, abdominal circumference, waist-pelvic circumference, body mass index Correspondence : Cindy Lestyani Loekito. Masters Program in Nutrition, Universitas Sebelas Maret. Jl. Ir. Sutami 36A, Surakarta 57126, Central Java. Email: cindy.l.l@student.uns.ac.id. Mobile:082134424950. Indonesian Journal of Medicine (2019), 4(3): 252-258 https://doi.org/10.26911/theijmed.2019.04.03.08

Recognition of rare hemoglobin variants by hemoglobin A1c measurement procedures

BackgroundUnrecognized hemoglobinopathies can lead to measured hemoglobin A1c (Hb A1c) concentrations that are erroneous or misleading. We determined the effects of rare hemoglobin variants on capillary electrophoresis (CE) and HPLC methods for measurement of Hb A1c. MethodsWe prospectively investigated samples in which Hb A1c was measured by CE during a 14-month period. For samples in which the electropherograms suggested the presence of rare hemoglobinopathies, hemoglobin variants were identified by molecular analysis or by comparison with electropherograms of known variants. When sample volume permitted, Hb A1c was measured by 2 HPLC measurement procedures and by boronate affinity HPLC. ResultsHb A1c was measured by CE in 33,859 samples from 26,850 patients. 15 patients (0.06%) were identified as having rare hemoglobinopathies: Hbs A2 prime, Agenogi, Fannin-Lubbock I, G Philadelphia, G San Jose, J Baltimore, La Desirade, N Baltimore, Nouakchott, and Roanne. Among 6 of these samples tested by 2 ion-exchange HPLC methods, the rare Hb was detected by both HPLC methods in only one sample, and none were detected by boronate affinity HPLC. The mean of the Hb A1c results of 2 HPLC methods differed from the result of the CE method by 0.7–2.2% Hb A1c in samples with variant hemoglobins versus <0.2% Hb A1c in samples without variants. ConclusionMeasurement procedures differ in the ability to detect the presence of rare Hb variants and to quantify Hb A1c in patients who harbor such variants.

The influence of sample freezing at - 80 °C for 2-12 weeks on glycated haemoglobin (HbA1c) concentration assayed by HPLC method on Bio-Rad D-10® auto analyzer.

IntroductionThe aim of the study was to evaluate the effect of a single freeze/thaw cycle on HbA1c concentrations measured by commercially available HPLC method.Materials and methodsStudy included 128 whole blood samples collected from diabetic patients (N = 60) and healthy volunteers (N = 68). HbA1c concentrations were measured in fresh blood samples. Then samples were frozen at - 80 °C for up to 12 weeks. HbA1c was assayed by ion-exchange HPLC method on Bio-Rad D-10® analyzer. Variables were compared using Wilcoxon and ANOVA Kruskal-Wallis tests. Bias between HbA1c measured in fresh and frozen samples was calculated. The comparability of HbA1c concentrations was assessed by Bland-Altman plot.ResultsMedian (IQR) HbA1c concentration was 45.3 (36.6–61.2) mmol/mol for fresh and 45.3 (36.6–60.6) mmol/mol for frozen/thawed samples. No significant difference in HbA1c concentrations was found comparing fresh and frozen/thawed samples (P = 0.070) in the whole group, as well as in healthy and diabetic subjects. The median calculated bias between fresh and frozen/thawed samples was 0% in whole group and healthy subjects, and 1.19% in diabetic patients. No significant difference was found between the biases according to baseline HbA1c values (P = 0.150). The Bland-Altman plot analysis showed a positive bias of 0.4% (95% CI: - 2.8 - 3.7%), which indicates high compliance between HbA1c values and no relevant influence of sample freezing on clinical significance of HbA1c measurement.ConclusionsStorage for up to 12 weeks at – 80 °C with a single freeze/thaw cycle does not affect HbA1c concentrations measured with HPLC method on Bio-Rad D-10® analyzer.

Association between Vitamin D, Fasting Blood Glucose, HbA1c and Fasting Lipid Profile in Euglycemic Individuals

In addition to biological actions of vitamin D such as calcium absorption, regulation of bone and mineral metabolism, studies have shown that Vitamin D is necessary for normal insulin secretion. Vitamin D influences insulin production and secretion through its effect on calcium and phosphorous metabolism. Vitamin D modifies insulin response by acting on receptor gene. Vitamin D deficiency has been implicated in decreased insulin secretion and increased insulin resistance. Objective: To measure and correlate vitamin D with fasting plasma glucose, HbA1c and fasting lipid profile in euglycemic individuals. Methods: Ethics clearance was obtained prior to data collection. After an overnight fast, blood samples were collected for fasting plasma glucose, lipid profile, HbA1c and vitamin D from euglycemic individuals who consented to participate. Vitamin D assay was estimated in Elecsys 2010 by ECLIA method. Plasma glucose was estimated by Hexokinase method. Serum fasting lipid profile was estimated in Cobas 6000 (Roche), and HbA1c measured using ion exchange HPLC method using Biorad Variant II turbo. Results: There was an inverse correlation between vitamin D and fasting plasma glucose (p＜0.001); Vitamin D and HbA1c (p＜0.001) in groups 1 and 3. There was also a significant inverse correlation of Total cholesterol (p=0.05), total cholesterol/HDL ratio (p＜0.001) with Vitamin D and significant direct correlation of HDL with vitamin D levels in groups 1 and 3. Conclusion: Deficiency of vitamin D can predispose to dysregulation of glucose and lipoprotein metabolism. There may be a role for vitamin D in better management of diabetes and dyslipidemia.

High HbA1C in anemic patients: Is hypovitaminosis D that link?

Context: Glycated hemoglobin is a form of hemoglobin that is measured primarily to identify the average plasma glucose concentration over prolonged periods of time, formed due to non-enzymatic glycation. Previous studies have suggested increased glycated hemoglobin levels in anemic patients despite the shortened life span of the erythrocytes. Several reports have suggested an active role of vitamin D in the functional regulation of pancreatic β-cells. Hypovitaminosis D may be an independent risk factor for type 2 diabetes mellitus (T2DM) with higher HbA1c levels. Studies have also demonstrate association of vitamin D deficiency and greater risk of anemia in children. Aims: The aim of this study is to estimate and analyse hemoglobin A1c, vitamin D and hemoglobin levels in non diabetic anemic adults and assess the relationship between above mentioned parameters. Materials and Methods: The study was conducted at Kasturba Medical College, Manipal. Serum vitamin D, hemoglobin and HbA1c measured in 187 non-diabetic anemic adults of 25-55 years of age, glycated hemoglobin measured in BIORAD variant turbo II using ion exchange HPLC method, and vitamin D by Eletrochemiluminescene method. Hemoglobin estimation was done by Drabkin's method. Results and Conclusion: The study found negative correlation between vitamin D and HbA1c in both males and females (r = -0.5, r = -0.4 P ≤ 0.001), HbA1c and hemoglobin (r = -0.4, r = -0.3 P ≤ 0.002). Also, there was significant positive correlation between the vit D and hemoglobin (r = 0.4, r = 0.3 P ≤ 0.001) values in the study population. Thus, there could be a role of vitamin D in the management of anemia. Since the sample size was small further study is recommended with larger sample size for reliable results.

Hemoglobin Seville [α 2 β 2 81(EF5) Leu → Phe] a silent phenotypic variant that interferes in hemoglobin A 1c measurement by ion-exchange HPLC method

Objectives The aim of the study was to investigate hemoglobin (Hb) species in a 61 year-old male with diabetes mellitus type II and a low value of Hb A 1c. Design and methods Hb species were analyzed by electrophoresis and chromatography methods. Functional properties were determined by oxygen equilibrium studies. β-globin gene was amplified by PCR and sequenced. Results and conclusions A novel clinically silent Hb (Hb Seville), that results in falsely low Hb A 1c measurement, was detected. This Hb variant presented a single base mutation at codon 81 (C → T) of the β-globin gene. This case points out the necessity of careful inspection of the chromatograms and the use of additional methods to Hb A 1c measurement when the presence of aberrant peaks is detected.

Effects of Hemoglobin (Hb) E and HbD Traits on Measurements of Glycated Hb (HbA1c) by 23 Methods

Glycohemoglobin (GHB), reported as hemoglobin (Hb) A(1c), is a marker of long-term glycemic control in patients with diabetes and is directly related to risk for diabetic complications. HbE and HbD are the second and fourth most common Hb variants worldwide. We investigated the accuracy of HbA(1c) measurement in the presence of HbE and/or HbD traits. We evaluated 23 HbA(1c) methods; 9 were immunoassay methods, 10 were ion-exchange HPLC methods, and 4 were capillary electrophoresis, affinity chromatography, or enzymatic methods. An overall test of coincidence of 2 least-squares linear regression lines was performed to determine whether the presence of HbE or HbD traits caused a statistically significant difference from HbAA results relative to the boronate affinity HPLC comparative method. Deming regression analysis was performed to determine whether the presence of these traits produced a clinically significant effect on HbA(1c) results with the use of +/-10% relative bias at 6% and 9% HbA(1c) as evaluation limits. Statistically significant differences were found in more than half of the methods tested. Only 22% and 13% showed clinically significant interference for HbE and HbD traits, respectively. Some current HbA(1c) methods show clinically significant interferences with samples containing HbE or HbD traits. To avoid reporting of inaccurate results, ion-exchange chromatograms must be carefully examined to identify possible interference from these Hb variants. For some methods, manufacturers' instructions do not provide adequate information for making correct decisions about reporting results.

Effects of 7 Hemoglobin Variants on the Measurement of Glycohemoglobin by 14 Analytical Methods

Hemoglobin variants (Hb(VAR)) are not uncommon in the Korean population, with Hb G-Coushatta and Hb Queens being the 2 most common Hb(VAR). Hb G-Coushatta is also the most common Hb(VAR) in Chinese people from the Silk Road region, as well as in some North American Indian tribes. However, data are scarce on the effect of these Hb(VAR) on the different methods used for analyzing HbA(1c). Specimens from 24 individuals with 7 Hb(VAR) (Hb G-Coushatta, Hb Queens, Hb G-Hsi-Tsou, Hb Ube-4, Hb G-Waimanalo, Hb Inglewood, and Hb Bologna-St.Orsola) were collected and tested using the International Federation of Clinical Chemistry primary reference method as well as 14 routine HbA(1c) assay methods. Hb G-Coushatta showed a clinically significant effect on the measured HbA(1c), particularly when analysis was performed with ion-exchange HPLC methods with short elution times. This interference could be resolved by measuring the HbA(1c) using other methods such as HPLC with a long elution time, immunoassay, boronate affinity chromatography, and enzymatic assay. Hb Queens showed a clinically significant difference, defined as a >10% deviation from regression lines, in results from the 2 HPLC methods but not in the other methods. The remaining 5 rare Hb(VAR) showed different HbA(1c) results in the different assays. Hb G-Coushatta, Hb Queens, and other rare Hb(VAR) can interfere with glycohemoglobin assays, including ion-exchange HPLC methods with short elution times, but the interference can be resolved using other unaffected methods. It is important to identify these Hb(VAR) through a careful inspection of the chromatograms and apply other noninterfering methods for accurate measurements of the HbA(1c).

Critical sample pretreatment in monitoring dried blood spot citrulline

Background Plasma Citrulline concentration has been correlated to functional enterocyte mass. Dried blood spot (DBS) analysis using LC-MSMS reduces sample amount needed. We optimized DBS elution to increase precision and accuracy of DBS LC-MSMS analysis. Method DBS control samples were eluted in varying pH (2.2–7.0) and for varying times (15–75 min) and Cit, Arg and Orn were analyzed using LC-MSMS, with and without derivatization. In 20 volunteers, the DBS LC-MSMS assay was correlated with a plasma ion exchange HPLC method. Results For Citrulline an extraction optimum was obtained at pH 2.6, whereas lower Arginine concentrations were found using low extraction pH. Increasing elution times lead to increased concentrations. Within-run CV was higher with, compared to without derivatization. No close association could be found between plasma HPLC and DBS LC-MSMS concentrations. Conclusion Analysis of amino acids on DBS using LC-MSMS should be optimized regarding the purpose of the assay. In our study, most optimal results were obtained without derivatization and elution in pH 2.6 for 45 min. Cellular amino acids in DBS might influence the correlation of Cit with severity of enteral disorders. Therefore, further evaluation of DBS Cit as a marker for enteral disorders is warranted.

Near-infrared spectroscopy (NIRS) with a fibre-optic probe for the prediction of the amino acid composition in animal feeds

The amino acids alanine, aspartic acid, glutamic acid, glycine, phenylalanine, valine, lysine, proline, and tyrosine present in feeds with different textures (blocks, tablets, granules and flour (meal) and used in different stages of animal feeding regimes (lactation, growth, maintenance, etc.) were analysed using near-infrared reflectance spectroscopy (NIRS) technology together with a remote reflectance fibre-optic probe. The method allows immediate control of the animal feeds without prior sample treatment or destruction through direct application of the fibre-optic probe on the sample. The regression method used was Modified Partial Least Squares (MPLS). The equations developed to determine the amino acid contents of the feeds afforded high values for the RSQ coefficient (0.814–0.963) in all the amino acids with the exception of lysine (0.687). The statistical prediction descriptors SEP, SEP(C) (with values between 0.134 for valine and 0.015 for aspartic acid) and bias indicated that the amino acid values in feeds predicted with NIRS with a fibre optic probe are comparable to those obtained with the chemical ion-exchange HPLC method.

Ion-exchange HPLC assay of tryptophan in breast milk of mothers with three months breast-feeding

Tryptophan is essential for the biosynthesis of biogenic amines. The correct ingestion of this amino acid is vital in the newborn for correct growth. An ion-exchange HPLC method is described for the assay of tryptophan in breast milk. The protein was hydrolysed by barium hydroxide and then assayed by the indicated chromatographic technique, establishing the conditions for the analysis. The values obtained confirmed the utility of the method for determining the tryptophan content in breast milk. The concentration of tryptophan found in the study population in mothers who had been breast-feeding for three months was 10.02 mgl−1 g−1 of protein.

The HPLC Separation and Bioactivity Evaluation of the Polysaccharopeptide (PSP) of Coriolus (Trametes) versicolor Fermentative Mycelia

Polysaccharopeptide (PSP, commercial name I'm-Yunity™) is isolated from the deep-layer cultivated mycelia of Coriolus versicolor Cov-1™ strain, using patented manufacturing processes. It is a covalently linked protein/polysaccharide complex with a molecular size reported as 100 kD. The polysaccharide moiety consists of 56.1% β-(1, 3) glycan. Its peptide moiety contains 18 kinds of amino acids in which the glutamic acid and aspartic acid are the most abundant. The pharmacological study and clinical trials have proved that PSP possesses immunomodulatory effects that can benefit cancer patients by improving their immunity and life quality. It also can antagonize the side-effects caused by radio- or chemotherapy. Previous studies have shown that PSP has several fractions. Electrophoresis results showed that PSP had three bands, and similar results were obtained using column chromatography. Yang MMP and colleagues separated PSP into two fractions by using reverse phase HPLC and detected the first eluant possessing direct antitumor activity. However, the contributions of all PSP fractions to its bioactivities are still not well understood. Therefore, in this study, we applied an anion ion-exchange HPLC method to separate PSP and evaluated the bioactivity of its fractions. The commercial PSP product was dissolved in 95 °C water and centrifuged to remove auxiliary materials and precipitants. It was further desalted and freeze-dried prior to HPLC separation. The freeze-dried powder was dissolved in an initial buffer and injected into a preparative anion ion-exchange HPLC column. The column was eluted with initial buffer following an increased NaCl gradient. After that, PSP was separated into four fractions: Fr1, without a negative charge; Fr2, with a weak negative charge; Fr3, with a moderate negative charge; and Fr4 with a strong negative charge. Chemical analysis reveals that the polysaccharide content of Fr2 is the highest (92.5%), while the Fr4 is the lowest (35.2%). However, the β-(1,3) glucan contents in polysaccharides are reversed, and the Fr4 has the highest content (51.0%), while Fr1 and Fr2 have almost no β-(1, 3) glucan detected. Fr1 and Fr2 also have roughly half-protein contents compared to Fr4 and Fr3, and the highest content is in Fr3 (22.6%). The mouse splenocyte proliferation assay reveals that the Fr4 and Fr3 have stronger immuno-stimulatory effects than Fr2 and Frl.The stimulatory index of Fr4, Fr3, Fr2, and Fr1 are 1.29, 1.27, 1.12, and 1.08, respectively. Fr3 and Fr4 also elevate mouse splenocyte CD4+/CD8+ ratio, while Fr1 and 2 retained or slightly decreased compared to unfraction-ized PSP. All four fractions strongly stimulate the production of IL-6 and TNF-α from human PBMN cells.The IL-6 production was increased up to around 8.1 fold by Fr 2 and 4, and TNF-α level was even heightened to 12.7 folds by Fr2 and Fr3. In conclusion, PSP probably contains several compounds, and these compounds can be fraction-ized using an anion ion-exchange HPLC method. Different fractions showed diverse pharmacological activities as well as different physical and chemical properties. To understand the relation of PSP fractions to their bioactivities, it may help us to set up specific quality control methods to monitor and to improve PSP quality and to develop the product with higher efficacy.

An isocratic ion exchange HPLC method for the simultaneous determination of flucloxacillin and amoxicillin in a pharmaceutical formulation for injection

An isocratic ion exchange high performance liquid chromatography method was developed for the simultaneous determination of flucloxacillin and amoxicillin in pharmaceutical formulations for injections. The separation was made by a ZORBAX 300-SCX column using 0.025 M ammonium dihydrogen phosphate (adjusted to pH 2.6 with phosphoric acid)–acetonitrile (95:5) as mobile phase. The validation of the method was performed, and specificity, reproducibility, precision and accuracy were confirmed. The limits of quantification were approximately 0.2 μg/ml for each drug. Due to its simplicity and accuracy the method is particularly suitable for routine pharmaceutical quality control.

Determination of glycated hemoglobin in clinically silent hemoglobin variants

Evaluation of glycated hemoglobin determination methods in patients with clinically silent hemoglobin variants. HbA1c results were determined with various methods, including a new enzymatic assay, a boronate affinity HPLC, immunoassays and ion-exchange HPLC in patients with the clinically silent hemoglobin variants Hb Graz, Hb Sherwood Forest, Hb D and Hb O Padova. The effect of hemoglobin variants on glycated hemoglobin determination was method-dependent. The enzymatic and boronate affinity HPLC method did not interfere with any of the variants evaluated. In contrast, Hb Graz interfered with all immunoassay and ion-exchange HPLC methods evaluated. The Tosoh ion-exchange HPLC method HLC-723 did not detect the late migrating Hb O Padova in the chromatogram, but this hemoglobin variant still interfered causing artificially low HbA1c results. Our study underscores the need for clinical laboratories and physicians to be aware of the limitations of their HbA1c assay methods as well as the importance of visual inspection of ion-exchange chromatograms to detect abnormalities caused by the hemoglobin variants. Samples with clinically silent Hb variants should be analyzed by a second method with a different assay principle, preferably a boronate affinity HPLC or an enzymatic assay.