Bioanalytical calibration curves: variability of optimal powers between and within analytical methods

The objective of this study was to investigate the variability of optimal power models in contrast to common regression models within and between analytical methods, as well as the frequency of outlier rejection. This was done by fitting the power model to calibration curve data using the minimum sum of squared residuals as a curve selection criterion. The jackknife percent deviation was used for detecting outliers. The data were obtained from 2087 analytical batches for 91 projects using various analytical techniques. The most frequent regression model varied between analytical techniques while the median and interquartile range of the optimal powers were stable. Outlier rejection is highest in GC and LCMS in which the Wagner (Quadratic, log-log) is the most frequent model. These results suggest that the greatest source of variability in the ideal transformation may not be the analytical technique but other within-lab sources. Outlying values may be due to these other sources of variability as suggested by the outlier rejection profile.     

Paramagnetic metal scavenging by melanin: MR imaging

To evaluate the potential application of high performance liquid chromatography or HPLC in performing hemoglobin typing, comparison between this technique and routine methods was carried out. Blood specimens from Pediatric Hematology Unit, Research Center, Faculty of Medicine, Ramathibodi Hospital were examined by these methods. The level of Hb A2, Hb E and Hb F were compared. Hb A2 level determined by HPLC and electrophoresis was statistically significantly different but correlated well. Good correlation was found between Hb A2 level from HPLC compared with microcolumn chromatography although there was a statistical difference. No difference but very good correlation was found between the level of Hb E from HPLC compared with electrophoresis. Statistical difference was encountered when Hb F level determined by HPLC was compared to that determined by Betke alkaline denaturation test. However, good correlation was observed when the level of Hb F was greater than 2.0%. In conclusion, HPLC could be an alternative way of performing hemoglobin typing provided that Hb F is 10% or more, by calculating from the equation: Alk F = (0.83 x Hb F by HPLC) - 0.98.     

Acetylcholine in human CSF: methodological considerations and levels in dementia of Alzheimer type

Four different methods to measure acetylcholine (ACh) and choline (Ch) concentration, i.e. thermospray/mass spectroscopy (TS/MS), high pressure liquid chromatography/mass spectroscopy (HPLC/MS), high pressure liquid chromatography/electrochemical detection (HPLC/ECD) and gas chromatography/mass spectroscopy (GC/MS), both latter methods coupled to a solid phase extraction system were compared for their applicability to human lumbar cerebrospinal fluid (CSF). Furthermore, samples from 15 control persons and 11 patients with dementia of Alzheimer-type (DAT) were compared to search for an ACh deficit in the CSF in DAT. GC/MS was the most sensitive, but most laborious method, and HPLC/ECD was acceptably sensitive, reliable and more specific. TS/MS was not specific enough for CSF extracts and HPLC/MS was more specific, but far less sensitive. Thus, only GC/MS and HPLC/ECD were qualified to detect ACh in human CSF extracts. Comparison of GC/MS and HPLC/ECD revealed highly correlated levels of ACh (r = 0.999). Using HPLC/ECD, ACh concentrations were greatly reduced in the DAT group (3.75 +/- 1.40 pmol/ml CSF) as compared to the controls (6.14 +/- 1.39 pmol/ml CSF), but the difference between controls and DAT patients was not statistically significant due to the number of samples below detection limit (8 out of 11 samples in DAT, 7 out of 15 in controls). Ch concentrations were not statistically significant between the two groups. The data show that methodological limitations preclude a widespread clinical application of determining ACh in the human CSF. Despite of reductions of ACh in the CSF in DAT, the determination of Ach in the CSF is not suitable for diagnostic purposes.     

Transmission and prevalence of Burkholderia cepacia in Welsh cystic fibrosis patients

EDTA-dependent pseudothrombocytopenia (PTCP) consists of an inappropriate low platelet count caused by autoantibodies present in the serum samples reacting with platelets only in EDTA-anticoagulated blood. By using immunoprecipitation and Western blot techniques, we studied the immunochemical specificity of platelet agglutinating autoantibodies in the serum samples of 10 patients with PTCP. Furthermore, to evaluate a possible role of PTCP-associated IgG autoantibodies in increased platelet turnover, we assayed the plasma glycocalicin (GC) level and calculated the GC index for every patient. Our results provide direct evidence that an epitope located on platelet membrane glycoprotein IIb is recognized by PTCP-associated IgG antibodies; moreover GC levels in patients with EDTA-dependent PTCP were similar to control levels, thus excluding an increased platelet turnover. We conclude that antiplatelet antibodies directed against platelet cryptantigens are unlikely to have a major role in the increased removal of cells from circulation.     

基于支持向量机和随机森林算法结合光纤式激光诱导击穿光谱定量检测核电用钢中铬

核电站关键设备的状态检测技术是核电安全性和经济性的重要保障,激光诱导击穿光谱技术(Laser-induced breakdown spectroscopy,LIBS)是一种可在线测量材料元素组分及其结构状态的新方法,基于采用光纤传输激光并回传等离子体辐射的光纤式激光诱导击穿光谱(fiber-optic LIBS,FO-...     

粉末压片制样-X射线荧光光谱法测定锂云母中铷、铯及主量组分

锂云母中铷、铯及主量元素的分析一般采用化学湿法,前处理过程复杂。实验采用粉末压片制样-X射线荧光光谱法(XRF)对锂云母中铷、铯及主量组分(二氧化硅、三氧化二铝、全铁、氧化钙、氧化钾、氧化钠)进行了测定。采用锂矿石标准物质和人工合成校准样品制作校准曲线,各组分的均方根为0.0042~0.49。校准曲线采用经验系数和康普顿散射线内标法校正组分间的吸收-增强效应,方法的检出限为3.1~188μg/g。按照实验方法测定锂矿石标准物质GBW 07152中铷、铯及主量组分,结果的相对标准偏差(RSD,n=10)在0.31%~5.0%之间。实验方法用于测定人工合成校准样品(未参与校准曲线的绘制)中铷、铯及主量组分,测定值与理论值吻合良好;测定2个锂云母实际样品中铷、铯及主量组分,测定结果与电感耦合等离子体原子发射光谱法(ICP-AES)的测定值相符。     

Determination of the International Sensitivity Index of a new near-patient testing device to monitor oral anticoagulant therapy--overview of the assessment of conformity to the calibration model

A key issue for the reliable use of new devices for the laboratory control of oral anticoagulant therapy with the INR is their conformity to the calibration model. In the past, their adequacy has mostly been assessed empirically without reference to the calibration model and the use of International Reference Preparations (IRP) for thromboplastin. In this study we reviewed the requirements to be fulfilled and applied them to the calibration of a new near-patient testing device (TAS, Cardiovascular Diagnostics) which uses thromboplastin-containing test cards for determination of the INR. On each of 10 working days citrated whole blood and plasma samples were obtained from 2 healthy subjects and 6 patients on oral anticoagulants. PT testing on whole blood and plasma was done with the TAS and parallel testing for plasma by the manual technique with the IRP CRM 149S. Conformity to the calibration model was judged satisfactory if the following requirements were met: (i) there was a linear relationship between paired log-PTs (TAS vs CRM 149S); (ii) the regression line drawn through patients data points, passed through those of normals; (iii) the precision of the calibration expressed as the CV of the slope was <3%. A good linear relationship was observed for calibration plots for plasma and whole blood (r = 0.98). Regression lines drawn through patients data points, passed through those of normals. The CVs of the slope were in both cases 2.2% and the ISIs were 0.965 and 1.000 for whole blood and plasma. In conclusion, our study shows that near-patient testing devices can be considered reliable tools to measure INR in patients on oral anticoagulants and provides guidelines for their evaluation.     

Evaluation of automated methods of measuring hemoglobin and hematocrit in horses

OBJECTIVE: To evaluate the accuracy of 3 automated methods of determining Hct and hemoglobin (Hb) concentration, compared with manual methods. Animals-22 clinically normal adult horses of various breeds. PROCEDURE: A blood sample was obtained from each horse. Six dilutions (representing Hct of 0, 10, 20, 40, 60, or 70%) were prepared from each sample and analyzed, using 1 of 2 blood gas analyzers or a hemoximeter (for automated determinations) or the Wintrobe macrohematocrit and cyanmethemoglobin methods (for manual determinations). Regression analysis was used to determine mean slope relationships between Hct and Hb measurements obtained by use of manual versus automated methods. Slopes were compared, using Student's t-test. RESULTS: Of the 3 automated methods examined, only 1 blood gas analyzer reported Hct and Hb values that were not significantly different from those determined by use of manual methods; however, this analyzer could not report Hb concentrations below 2.5 g/dl. The other blood gas analyzer reported values for Hct and Hb concentrations that were consistently higher than those obtained by use of manual methods at Hct < or = 20% and Hb < or = 6.6 g/dl. The hemoximeter yielded more accurate results if the Hb concentration was between 6.6 and 20 g/dl. CONCLUSION: Although there were some limitations in measuring at low Hb concentrations, the method of determining Hb concentration and Hct with blood gas analyzer 2 was more accurate than that with blood gas analyzer 1 (Hct and Hb concentration) or the hemoximeter (Hb only).     

Cranial vault moulding by the transcutaneous activation of implanted magnets

The present paper surveys the published HPLC methods evaluating profens in biological materials and reports a HPLC method for the determination of ibuprofen in the samples of whole blood. For liquid-liquid extraction, ibuprofen was analyzed on the reverse phases Separon SGX C18 with the mobile phase methanol-water (pH 3.0) and detected at 222 nm. The elaborated method was employed in a pharmacokinetic study of ibuprofen on laboratory rabbits.     

粉末压片-X射线荧光光谱法测定含铌多金属矿样中铌

采用粉末压片制样,使用X射线荧光光谱仪对含铌多金属矿样中的铌进行测定。由于含铌的多金属矿标样极少且含量较低,实验选取钽矿石标准样品、矿区具有代表性的化学法定值多个样品,及其他土壤、岩石、多金属矿物标准样品、矿区定值样品混合配制的校准样品,制成一套铌含量5.9～2 700 μg/g、梯度适当的校准样品系列,绘制的铌校准曲线相关系数为0.998 6。采用经验系数和康普顿散射线内标法校正了基体效应,用Omnian 近似定量软件、化学分析法与岩矿鉴定分析,确定了矿区矿物中主要成分SiO₂、Al₂O₃、CaO、MgO、K₂O、Pb、Zn、Cu、Fe、Zr、Mo、Rb、Hf、Th、U、Ti、Ga及稀土的最高允许量。综合考虑样品基体对铌检出限的影响,实验选取8个标准样品计算出检出限的平均值为1.62 μg/g。对岩石标样进行精密度考察,结果的相对标准偏差(RSD,n=12)为2.2%。对标准样品及矿区实际样品进行分析,测定值与认定值、实验室内其他方法的测定值及其他实验室的测定值吻合,满足《地质矿产实验室测试质量管理规范》的要求。     

Development of a densitometric method for the determination of cephalexin as an alternative to the standard HPLC procedure

A HPTLC-densitometric method was developed in order to obtain a reliable procedure for routine analysis of cephalexin in pharmaceutical formulations. Optimization of TLC conditions for the densitometric scanning was reached by eluting HPTLC silica gel plates in an horizontal developing chamber. Quantitation of cephalexin was performed in single beam reflectance mode by using a computer-controlled densitometric scanner and applying a five-point calibration. A linear regression has been found in the 200-1000 ng range. The setup method is precise, reproducible and accurate. Recovery was also assessed by comparison with the HPLC USP XXIII alternate method. In this case HPTLC-densitometry appears worth of consideration as being relatively inexpensive and time-saving (up to 12 samples can be determined simultaneously in less than 15 min with a solvent consumption of about 15 ml). The results suggest that the proposed method may be used in place of HPLC for the routine quantitation of cephalexin in both pure and dosage forms.     

Quantification of orotic acid in dried filter-paper urine samples by stable isotope dilution

A rapid, sensitive, and specific method for quantification of orotic acid from dried filter-paper urine samples is described. The method involves stable isotope dilution with 1,3-[15N2]orotic acid analysis by gas chromatography-mass spectrometry. The assay is sufficiently sensitive to be used with solvent extraction techniques commonly used for urinary organic acid analysis. Extraction efficiencies of both native and isotopic orotic acid from dried filter paper and from water were 31% and 28%, respectively. The concentration of orotic acid in dried filter-paper urine specimens from 50 healthy controls was 1.1 +/- 0.67 (mean +/- SD) mmol/mol of urinary creatinine. The same 50 urine samples, analyzed directly from a 5-mL aliquot of liquid urine, gave values of 0.93 +/- 0.51. The correlation coefficient between the results obtained by the two different collection methods was 0.87. Age-related reference values in filter-paper samples are also reported. The concentrations, which are normalized to urinary creatinine, decrease with age. This method is applicable to rapid screening for urea cycle disorders and may also be used for carrier testing of ornithine transcarbamylase deficiency.     

熔融制样-X射线荧光光谱法测定锆质耐火材料中11种主次组分

标准方法GB/T 4984—2007对于含锆耐火材料中主次组分的测定需要多种方法相结合,较为繁琐。采用选择日本耐火材料协会X射线荧光光谱分析专用锆质耐火材料系列标样和ZrO₂基准试剂混合配制2个校准样品,用该系列标准样品和校准样品制备出12个标准样品/校准样品玻璃片,建立了测定锆质耐火材料中11种主次组分(ZrO₂、HfO₂、Cr₂O₃、MgO、CaO、SiO₂、Al₂O₃、Fe₂O₃、TiO₂、K₂O、Na₂O)的X射线荧光光谱分析方法。实验表明,控制称样量为0.400 0 g,采用Li₂B₄O₇-LiBO₂(m∶m=12∶22)混合熔剂和1∶15的稀释比,加入0.2 mL 300 g/L NH₄I溶液为脱模剂,于1 050 ℃下熔融15 min,熔出的样片均匀光滑,达到检验要求。选择ZrLɑ和HfLβ1线为分析谱线,克服了ZrKɑ谱线荧光强度过高的影响;通过谱线校正消除谱线干扰,依据经验α系数法进行基体校正,扣除了样品中各元素的吸收增强效应。校准曲线精密度品质因子小于0.07,各组分检出限为0.007%～0.024%之间。采用实验方法对锆质耐火材料样品进行检测,各组分测量结果的相对标准偏差(RSD)在0.24%～4.9%之间。选择由专用锆质耐火材料系列标准样品和ZrO₂基准试剂混合配制的合成样品,分别采用实验方法和标准方法GB/T 4984—2007或GB/T 6900—2016对11种组分进行分析,发现实验方法和标准方法测定值保持一致,实验方法与参考值的误差在标准方法允许差范围内。     

Evaluation of multiwavelength chromatograms for the quantification of mixtures of pesticides by high-performance liquid chromatography-diode array detection with multivariate calibration

Three multivariate calibration methods, partial least squares (PLS-1 and PLS-2) and principal component regression, were applied to the simultaneous determination of the five pesticides iprodione, procymidone, chlorothalonil, folpet and triazophos by high-performance liquid chromatography with diode array detection. Such detection gives multiwavelength chromatograms from a single analysis of one sample. In this paper, calibration models at two different wavelengths were developed to resolve mixtures of five pesticides with overlapping chromatographic peaks. The first model, carried out at 220 nm as detector compromise wavelength, yielded satisfactory sensitivity for accurate estimation of the concentration of iprodione, procymidone, chlorothalonil and folpet and the second model, at 200 nm, was used for accurate estimation of triazophos. Both calibration models were evaluated using the chromatograms and first-derivative (1D) chromatograms by predicting the concentrations of independent test set samples. Finally, the proposed 1D calibration models were successfully applied to the determination of these pesticides in groundwater and soil samples. In all cases, the PLS-1 calibration method showed superior quantitative prediction ability than the PLS-2 or principal component regression methods.     

金属标准物质中杂质的辉光放电质谱法溯源分析

标准物质定值分析的溯源性要求比常规分析高很多。在常规分析中,有证标准物质（CRM）可用于校准以及建立可溯源性。尽管与标准物质生产和认定相关的ISO导则并没有十分明确地提到校准,但众多标准物质生产者在定值分析时却不接受基准标准物质用于校准。这些要求制约了标准物质定值方法的使用,因为这些方法可以采用已知高纯度的标准物质、化学计量法及已知高纯度标准物质的混合物进行校准。有效的固体取样技术,例如通常使用的块状标准物质进行校准的辉光放电质谱法（GD MS）或火花源发射光谱法（spark OES）,似乎不太适合定值分析。尤其是固体样品中痕量元素分析测定的有力工具辉光放电质谱法,除金属杂质外,使用特定气体混合物,这种方法也可以分析相关的非金属杂质如硫和磷。我们开发了一种可以使用已知高纯度的标准物质混合物和化学计量法的校准方法,这种校准方法同样可用于辉光放电质谱法。与溶液中基体匹配技术类似,此校准方法基于掺杂的粉末压片。本文还结合铜和钢标准物质的定值分析结果进行了陈述。     

Determination of hepatotoxic pyrrolizidine alkaloids by on-line high performance liquid chromatography mass spectrometry with an electrospray interface

The present study describes the determination of two different types of hepatotoxic pyrrolizidine alkaloids (PAs) and also distinguishing the hepatotoxic PAs from non-toxic ones by both in-source collision-induced dissociation high performance liquid chromatography mass spectrometry (CID-HPLC/MS) and HPLC/MS/MS (CID in the collision cell), using electrospray ionization. The mass spectra provided molecular ions and characteristic fragment ions, which could be used readily for a rapid identification of different types of PAs. Applications of both in-source CID-HPLC/MS and HPLC/MS/MS analytical methods were successful for the determination of PAs in blood samples obtained from rats dosed with PAs and in the PA-containing plant. The results demonstrated that the developed HPLC/MS methods with two different CID techniques provided a very simple and rapid analysis for an unequivocal diagnosis of PA poisoning and for definitive identification of PAs in plants or herbal medicines.     

Protein interactions during assembly of the enamel organic extracellular matrix

This paper describes a simple and sensitive high-performance liquid chromatographic (HPLC) method for the detection of human globin chains in blood and bloodstains. The method involves direct injection of the filtered samples of dilute hemolysates or bloodstain extracts onto a microbore C4 reversed-phase column (2.1 mm I.D.) with UV detection at 220 nm. Microbore HPLC offers a significant improvement in sensitivity with little loss of the resolution of globin chains and only small variations in the determination of gamma chain composition. The detection limit of hemoglobin (Hb) was 0.1 microgram, which is equivalent to about 1 nl of fresh whole blood. Umbilical cord blood could be differentiated from adult blood in stains that were up to twenty weeks old, by the presence of gamma globin chains. The present method will be useful for detection of abnormal Hbs and for the determination of gamma chain composition in clinical laboratories, as well as in the practice of forensic science for the analysis of minute amounts of blood and bloodstains.     

Prenatal and postnatal diagnoses of thalassemias and hemoglobinopathies by HPLC

The conventional approach to qualitative and quantitative analyses of hemoglobin (Hb) molecules for the diagnoses of hemoglobinopathies requires a combination of tests. We used an automated HPLC (VARIANT) system to study alpha-thalassemia and beta-thalassemia syndromes in Thailand. The beta-thalassemia short program is applicable to the diagnosis of alpha-thalassemia and beta-thalassemia disorders, including Hb H, EA Bart's disease, and EF Bart's disease, in adults, newborns, and fetuses. The system cannot quantify accurately certain Hb molecules, such as Hb H and Hb Bart's. The alpha-thalassemia short program was therefore developed and used to quantify Hb Bart's to detect alpha-thalassemia genotypes in cord blood. This automated HPLC system is an alternative approach to the diagnosis of complicated thalassemia syndromes in Thailand and Southeast Asia.     

Mechanism of 17 alpha-hydroxylase/17,20-lyase--an initial geometric perspective for the lyase of the C(17)-C(20) bond of C21 steroids

A biosensor based on an H+ ion sensitive field effect transistor (H+(-)ISFET) and penicillin G acylase has been developed. The response time of the sensor to different concentrations of penicillin G was 30 s. In a 20 mM phosphate buffer at pH 7.0, the linear range of the calibration curve was from 0.5 to 8 mM. The coefficients of variation for three samples with 20 repeated measurements were below 5%. Stability of the sensor could reach about 6 months and more than 1000 runs were performed without a significant decrease of the output value. The sensor was tested for measurement of the penicillin G content in penicillin fermentation both. Forty samples with low and high concentrations of penicillin G were chosen for the correlation test. The values assayed by the sensor method were compared with the values assayed by HPLC method, the correlation coefficient (r) was 0.9944 and the regression equation was y = 1.034X - 2083.7, respectively. The different measuring methods are discussed in the text.     

Analysis of retinoic acid receptor beta expression in normal and malignant laryngeal mucosa by a sensitive and routine applicable reverse transcription-polymerase chain reaction enzyme-linked immunosorbent assay method

We have developed two assays for complete analysis of hemoglobins (Hbs) in the field of hemoglobinopathies: a high-performance cation-exchange liquid chromatography (HPLC) assay on the weak cation-exchanger Poly Cat A and a two-step capillary isoelectric focusing (CIEF) assay on the neutral-coated capillary from Beckman in a narrow pH gradient. The resolution was satisfactory for both HPLC and CIEF and allowed separation of normal and common abnormal Hbs, i.e., Hb A, Hb F, Hb A2, Hb S, Hb C, and Hb E; slight differences were shown for the resolution of unusual variants such as Hb C-Harlem and Hb D-Punjab. The reproducibility of retention times was satisfactory as well for HPLC (CV 3.3%) and CIEF (CV 4.9%). The imprecision of quantification of Hb A2, evaluated at two concentrations, and of Hb F and Hb S was < 5%, except for low concentrations of Hb A2 quantified by CIEF. Quantitative data obtained for these three Hb forms were highly correlated between the two assays. These results suggest that the new CIEF assay can be competitive with HPLC for complete routine analysis of Hb variants.