Tocopherol Acetate Reference Standard (Control 941) of the National Institute of Health Sciences

The raw material of betamethasone was tested for preparation of the "Betamethasone Reference Standard (Control 951)". Analytical data obtained were as follows: loss on drying, 0.0%; infrared spectrum, the same as that of the JP Betamethasone Reference Standard (Control 845); thin-layer chromatography, no impurity was detected; high-performance liquid chromatography (HPLC), two kinds of impurities were detected and the total amount was estimated to be 0.16 +/- 0.01% (n = 3); assay, 100.0% by HPLC. Based on the above results, the raw material was authorized as the JP Betamethasone Reference Standard (Control 951).     

The Cyclandelate Reference Standard (Control 931) of the National Institute of Health Sciences

Raw thiamine hydrochloride material was tested for preparation of the "Thiamine Hydrochloride Reference Standard (Control 931)". Analytical data obtained were as follows: melting point, 242.7 degrees C (decomposition); infrared spectrum, the same as that of the JP Thiamine Hydrochloride Reference Standard; thin-layer chromatography, one impurity was detected; high-performance liquid chromatography (HPLC), a trace amount of one impurity was detected; assay results, 100.4% by UV spectrophotometry and 100.0% by HPLC, respectively. Based on the above findings, the raw material was authorized as the JP Thiamine Hydrochloride Reference Standard (Control 931).     

Riboflavin Reference Standard (Control 921) of National Institute of Health Sciences

The raw material for riboflavin was tested for preparation of the "Riboflavin Reference Standard (Control 921)". Analytical data obtained were as follows: melting point, 283.8 degrees C (decomposition); specific absorbance, E1%1 cm = 852 (267 nm), 275 (373 nm), 325 (446 nm); infrared spectrum, the same as that of JP Riboflavin Reference Standard; optical rotation [alpha]20D = 138.5 degrees; thin-layer chromatography, no impurities were detected up to 6 micrograms; high-performance liquid chromatography, a small amount of 9 impurities were detected; loss on drying, 0.16%; assay, 100.3% by spectrophotometry. Based on the above results, the raw material was authorized as the Japanese Pharmacopoeia Reference Standard (Control 921).     

Hydrocortisone Acetate Reference Standard (Control 961) of National Institute of Health Sciences

The raw material of hydrocortisone acetate was tested for the preparation of the "Hydrocortisone Acetate Reference Standard (Control 961)". Analytical data obtained were as follows: IR spectrum, specific absorption wave numbers at 3428, 1748, 1723, 1631, and 1375 cm-1; specific absorbance, E1cm1% (242 nm) = 406; thin-layer chromatography, no impurities were detected until 0.05 microgram; high-performance liquid chromatography (HPLC), 2-3 impurities were detected and the amount of the total impurities was estimated to be about 0.2%; assay by HPLC, 100.7%. Based on the above results, the raw material was authorized as the Japanese Pharmacopoeia Reference Standard (Control 961).     

Gemfibrozil enhances platelet activity in patients with combined hyperlipoproteinemia

We have examined the pharmacokinetic interaction between isradipine and lovastatin in six male and six female, healthy, normotensive, human subjects after a single dose and after treatment for 5 days. The isradipine plasma concentrations were determined by a radioimmunoassay and the lovastatin serum concentrations by gas chromatography-mass spectrometry (GC/MS) and by the inhibition of the 3-hydroxy-3-methylglutaric-coenzyme reductase activity. We found that the apparent serum concentrations of lovastatin were 4- to 6-fold higher in the reductase-inhibition assay than the GC/MS assay, suggesting that the bulk of the reductase inhibition is due to active metabolites. The peak and the time-to-peak concentrations were unaffected by the treatments, either after the first dose or after continued administration. In male subjects, after repeated doses of isradipine, the lovastatin area under the time-concentration curves (AUCs) decreased by 40% as determined by the GC/MS assay (P < .001) and 20% as determined by the reductase-inhibition assay (P < .0022). In the female subjects, isradipine treatment decreased the lovastatin AUCs as determined by the GC/MS assay, but this was not statistically significant due to a high variance. Furthermore, in the female subjects, isradipine had no effect on the lovastatin AUCs as determined by the reductase-inhibition assay. Because the lovastatin peak and the time-to-peak concentrations were unaffected by isradipine treatment, the decreased lovastatin AUCs were probably not due to altered intestinal absorption. More likely, because lovastatin has a high hepatic clearance, the decreased AUCs seen after isradipine treatment could be due to increases in the clearance of lovastatin secondary to increased hepatic blood flow.     

Polymorphism of human pituitary FSH: analysis of immunoreactivity and in vitro bioactivity of different molecular species

Follicle-stimulating hormone is known to be highly heterogeneous in serum and in the pituitary. In the present study, we have partially separated different molecular species of human pituitary FSH and characterized their immunoreactivity and in vitro bioactivity. Pooled extracts of male (n = 15) and female (n = 9) human pituitary glands were chromatographed on a column of Sephacryl S-200 and FSH-containing fractions were fractionated by chromatofocusing in the pH range 4-6. FSH was measured in the individual fractions by an in vitro bioassay, based on the FSH-dependent aromatase activity of immature rat Sertoli cells, and by the following methods based on commercial kits: radioimmunoassay (RIA), immunofluorimetric assay (IFMA), immunoradiometric assay (IRMA), immunoenzymometric assay (IEMA). In each assay, the kit standard, calibrated against the 2nd International Reference Preparation (IRP) 78/549, and the International Standard (IS) 83/575 were run in parallel. The relative potencies of the kit standards in terms of IS 83/575 were: IFMA 3.08, IRMA 1.62, RIA 2.42, IEMA 1.45 and bioassay 1.14. After chromatofocusing, pituitary FSH eluted mostly in fractions with pH approximately 4.5, without sex-related differences. In both sexes approximately 25% of bioactive material showed a pI < 4 and eluted with 1 M NaCl. Although the same IS 83/575 was used in the various assays, the profiles of immunoreactive FSH were significantly different. The highest intermethod variability was observed in the case of male pituitary FSH. The relative biopotency of the different molecular species of FSH did not appear to change according to their pI but, rather, varied with the assay method and the standard.(ABSTRACT TRUNCATED AT 250 WORDS)     

Capillary isoelectric focusing and sodium dodecyl sulfate-capillary gel electrophoresis of recombinant humanized monoclonal antibody HER2

Capillary isoelectric focusing (cIEF) and IEF of recombinant humanized monoclonal antibody HER2 (rhuMAbHER2) show five charged isoforms with estimated pI values ranging from 8.6-9.1. The cIEF assay demonstrated good precision with relative standard deviations (R.S.D.) 0.7-3.7% and 0.4-4.2% for intra and interassay analysis, respectively. The method was linear for the area of the main peak over the concentration range 2-250 micrograms/ml with a Pearson correlation coefficient > 0.99. The limit of detection for the main peak was determined to be 2 ppm. With both sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) and SDS-polyacrylamide gel electrophoresis, the nonreduced rhuMAbHER2 migrated as a single major peak with minor peaks in the aggregate and clip regions. After reduction, the electropherogram and the slab gel showed the expected heavy chain and light chain fragments with minor peaks in the aggregate and clip regions. The SDS-CGE assay showed good precision with R.S.D. values of 0.1-7.8% and 0.1-8.1% for intra and interassay analysis, respectively. The Pearson correlation coefficient for the area of the main peak was > 0.99 demonstrating linearity for the concentration range 0.5-500 micrograms/ml. The limit of detection for intact rhuMAbHER2 was determined to be 0.5 ppm. The data presented demonstrates the feasibility of replacing the slab gel techniques with capillary electrophoresis in a quality control environment.     

冶炼烟气硫酸净化工艺对制取试剂硫酸的影响研究

利用某铅锌冶炼厂冶炼烟气硫酸为原料,经分析化验,原料酸中杂质含量较高,直接蒸馏对试剂硫酸的质量影响较大。因此,在蒸馏前需对原料酸进行净化处理,对原料酸的净化工艺进行了试验研究,采用经过净化的原料酸制取的试剂硫酸产品指标达到国家标准GB/T625-2007化学纯指标要求。     

Structural dynamics of the Streptomyces lividans K+ channel (SKC1): oligomeric stoichiometry and stability

SKC1, a 160-residue potassium channel with two putative transmembrane (TM) segments was recently identified from Streptomyces lividans. Its high levels of expression, small size, and ease of purification make SKC1 an ideal candidate for high-resolution structural studies. We have initiated the structural characterization of this channel by assessing its oligomeric behavior, stability in detergent, general hydrodynamic properties, and preliminary secondary structure content. SKC1 was readily expressed and purified to homogeneity by sequential metal-chelate and gel filtration chromatography. Standard SDS-PAGE, together with chemical cross-linking analysis indicated that SKC1 behaves as a tightly associated tetramer even in the presence of SDS. Using a gel shift assay to assess its oligomeric state, we determined that SKC1 is stable as a tetramer in most detergents and can be maintained in nonionic detergent solutions for extended periods of time. The tetramer is also stable at relatively high temperatures, with an oligomer-to-monomer transition occurring at approximately 65 degrees C. The Stokes radius of the micellar complex is 5 nm as determined from gel filtration chromatography of SKC1 in dodecyl maltoside. Preliminary estimations of secondary structure from CD spectroscopy showed that the channel exists mostly in alpha-helical conformation, with more than 50% alpha-helical, close to 20% beta-sheet, 10% beta-turn, and about 15% unassigned or random coil. These results are consistent with the idea that a bundle of alpha-helices forming a tetramer around the ion-conductive pathway is the common structural motif for members of the voltage-dependent channel superfamily.     

峰面积修正-X射线荧光光谱法测定铂首饰中铂

Pt与Au的原子序数相近,X射线激发下,影响它们荧光强度的诸多因素都非常相近,据此建立了利用金首饰系列标准物质(只含Au、Cu元素)和Pt、Cu峰面积修正,X射线荧光光谱法(XRF)测定铂首饰样品(只含Pt、Cu元素)中Pt含量的分析方法。首先,通过测定金首饰系列标准物质,绘制Cu含量(w_Cu)与Cu、Au峰面积比值Q(I_Cu/I_Au)之间的校准曲线;其次,通过试验对铂首饰标准物质的Pt和Cu的峰面积进行修正,并计算其Q值;最后,采集铂首饰样品的谱图,求Q值,查校准曲线,即可求出铂首饰中的Cu含量,进而间接计算出Pt的含量。方法适用于铂质量分数为91.70%～99.92%的铂首饰的测定。对5件铂首饰样品进行测定,结果与原子吸收光谱法(AAS)一致。实验方法解决了测定铂首饰中的铂时缺乏铂标准样品的难题。     

Automatic accurate non-invasive quantitation of blood flow, cross-sectional vessel area, and wall shear stress by modelling of magnetic resonance velocity data

OBJECTIVES: To apply a new, automatic and non-invasive method for quantification of blood flow, dynamic cross-sectional vessel area, and wall shear stress (WSS) by in vivo magnetic resonance velocity mapping of normal subjects. DESIGN: Prospective, open study. MATERIALS: Six young volunteers. METHODS: A three-dimensional paraboloid model enabling automatic determination of blood flow, vessel distensibility and WSS was applied to blood velocity determinations in the common carotid artery. Blood flow was also determined by a manual edge detection method. RESULTS: Using the new method, the common carotid mean blood flow was 7.28 (5.61-9.63) (mean (range)) ml/s. By the manual-method blood flow was 7.21 (5.55-9.60) ml/s. Mean luminal vessel area was 26% larger in peak systole than in diastole. Mean/peak WSS was 0.82/2.28 N/m2. Manually and automatically determined flows correlated (r2 = 0.998, p < 0.0001). WSS and peak centre velocity were associated (r2 = 0.805, p < 0.0001). CONCLUSIONS: Blood flow, luminal vessel area dilatation, and WSS can be determined by the automatic three-dimensional paraboloid method. The hypothesis of association between peak centre velocity and WSS was not contradicted by the results of the present study.     

Determination of calcium by inductively coupled plasma-atomic emission spectrometry, and lead by graphite furnace atomic absorption spectrometry, in calcium supplements after microwave dissolution or dry-ash digestion: method trial

A 3-laboratory method trial was conducted to evaluate 2 sample digestion procedures and instrumental determination parameters for analysis of calcium and lead in Ca supplements. Calcium supplements were treated by dry-ash digestion or microwave dissolution prior to spectrometric analysis. In each case, Pb was determined by graphite furnace atomic absorption spectrometry and Ca by inductively coupled plasma-atomic emission spectrometry. Blind duplicates of 6 Ca supplement samples were analyzed after each sample treatment procedure. Matrix pairs contained dissimilar Pb levels to cover the analyte range encountered during method development. Calcium content of the Ca supplement samples also reflected the range seen during method development. Stock solutions of Ca and Pb were supplied to collaborators for preparation of quantitation standards to remove a variable external to the method. National Institute of Standards and Technology Standard Reference Material (NIST SRM) 1486, bone meal, was included to assess method accuracy and recovery at NIST certificate Ca and Pb levels for this material (26.58 +/- 0.24% Ca and 1.335 +/- 0.014 micrograms Pb/g). Analyses of the NIST SRM yielded 25.9 +/- 1.1 and 27.2 +/- 2.3% Ca and 1.53 +/- 0.19 and 1.26 +/- 0.19 micrograms Pb/g for dry-ash and microwave procedures, respectively. Statistical analyses of data indicated acceptable repeatability and reproducibility for determination of Pb and Ca in various Ca supplements. With either sample preparation technique, the method is appropriate for determining Pb or Ca in Ca supplements.     

铅试金-石墨炉原子吸收光谱法测定铂钯矿浸出液中铂和钯

铂钯矿浸出液中铂和钯的准确测定,对铂和钯的综合利用具有十分重要的意义。铂钯矿浸取液中铂和钯含量较低,且还含有大量的共存离子,若直接对铂和钯进行测定,干扰较为严重。采用铅试金法对样品中铂和钯进行分离富集,在优化仪器参数的基础上,建立了石墨炉原子吸收光谱法(GF-AAS)测定铂钯矿浸出液中铂和钯的新方法。将10 mL铂钯矿浸出液滴在试金配方凹槽内,滴入硝酸银溶液作为灰吹保护剂,再覆盖试金配方,经高温熔融和灰吹,样品中的铂和钯被富集于银合粒中。采用先加入硝酸、再加入盐酸的方式溶解银合粒,用石墨炉原子吸收光谱法进行测定,实现了对样品中铂和钯的测定。优化了铂和钯的石墨炉灰化温度和原子化温度以及原子化读数时间。在选定的优化实验条件下,铂和钯的吸光度与其对应的质量浓度运用二次方程最小二乘法拟合校准曲线,曲线拟合良好,铂和钯校准曲线的决定系数分别为0.999 8、0.999 7,特征浓度分别为2.14、0.34 ng/mL。将实验方法应用于铂钯矿浸出液中铂和钯的测定,测得结果的相对标准偏差(n=6)为2.7%～5.7%,加标回收率为84%～118%,加标回收率满足国家地质矿产行业标准DZ/T 0130—2006的要求。     

The hereditary hyperbilirubinaemias

A capillary isoelectric focusing (cIEF) method has been developed for the purpose of determining the identity and charge distribution of mouse/human chimeric antibody to human CD20 antigen (C2B8). The assay was validated in accordance with ICH guidelines in order to demonstrate that it is suitable for its intended purpose and so that it may be performed as a lot release test for bulk and final product. As a result of the validation process the assay was found to be linear over the concentration range of 2-356 micrograms ml-1 with recovery of 125I-labeled C2B8 at the target sample concentration of 125 micrograms ml-1 equal to 99%. The repeatability and intermediate precision relative standard deviations of the four major peaks for migration time, peak area, and peak area percent ranged from 0.9-4.4%. The specificity of the assay was demonstrated by baseline resolution of the C2B8 main peak from product excipients, and other Genentech monoclonal antibodies. The results of this validation demonstrate that the cIEF assay for the determination of identity and charge distribution of C2B8 is accurate, precise, linear, and highly specific. The assay is rapid and suitably rugged.     

Estrogen and progesterone receptors in endometrial carcinoma: comparison of immunohistochemical and biochemical analysis

In 159 endometrial carcinomas, estrogen (ER) and progesterone receptors (PR) were determined biochemically by dextran-coated charcoal (DCC) assay and immunohistochemically (ICA) on frozen sections. ICA receptor content was estimated by a total histologic score (HSCORE), including all tissue components, and by a cancer HSCORE, including malignant cells only. These scores were closely correlated. A single biopsy was found to be representative for each tumor. ER-DCC status was positive in 90.3% and PR-DCC status in 92.2% of the tumors. ER total HSCORE was positive in 47% and PR total HSCORE in 89% of tumors. ER and PR correlated inversely with tumor grade (p < 0.001). Correlations were found between ER and PR content determined by either method (DCC: r = 0.77; ICA: r = 0.50), as well as between DCC and ICA content (ER: r = 0.52; PR: r = 0.76). The association between DCC and ICA was affected by the tumor grade: the DCC values decreased relatively more than total HSCOREs with increasing grade. The sensitivity of ICA against DCC assay was 56% for ER and 86% for PR. Maximal agreement between receptor status as determined by ICA and by DCC would result from a DCC cutoff level of 130 fmol/mg for ER and 114 fmol/mg for PR.     

高频燃烧红外吸收法测定铜精矿中硫的助熔剂探讨

称取0.040 0 g试样,将试样置于灼烧过并铺有0.3 g五氧化二钒助熔剂的坩埚内,加入0.2 g锡粒,再覆盖0.15 g五氧化二钒助熔剂和 1.4 g钨粒,以硫酸钾绘制校准曲线,建立了高频燃烧红外吸收法测定铜精矿中硫质量分数为5.00%～40.00%的方法。实验表明,以积分面积为横坐标,硫绝对含量为纵坐标绘制校准曲线。校准曲线的线性方程为Y=37.02X－1.52,线性相关系数R=0.999 8。方法检出限为0.017%。采用实验方法对铜精矿实际样品中硫含量进行测定,所得结果与重量法或燃烧-滴定法基本一致。采用实验方法对铜精矿标准样品进行测定,测定值与认定值基本一致。对铜精矿实际样品和标准样品6次平行测定结果的相对标准偏差(RSD,n=6)为0.41%～0.72%。     

How much insulin-like growth factor I (IGF-I) circulates? Impact of standardization on IGF-I assay accuracy

There is a significant systematic difference between the normal range obtained from ethylenediamine tetraacetate plasma samples using the Genentech total insulin-like growth factor I (IGF-I) RIA and normal ranges for other total IGF-I RIAs. To determine whether the quality of the assay standard was the cause of this systematic difference, we analyzed commercially available preparations of recombinant human IGF-I (rhIGF-I) typical of those used as IGF-I immunoassay standards along with our own well characterized rhIGF-I assay standard. For the commercial standards, high performance liquid chromatography-derived purities were low, and some vendor-assigned protein concentrations were inconsistent with values from quantitative amino acid analysis. The Genentech rhIGF-I assay standard was highly pure and quantitatively correct. However, the poor quality of some commercial rhIGF-I preparations was not the primary reason for the systematic discrepancy between the Genentech total IGF-I RIA normal range and most other normal ranges. Most assays for total IGF-I are calibrated against the WHO International Reference Reagent (IRR) for IGF-I Immunoassays (87/518). The Genentech total IGF-I RIA is not calibrated against WHO IRR 87/518. The protein content assigned to WHO IRR 87/518 was a consensus value from a multicenter collaborative study. Physicochemical analyses showed that WHO IRR 87/518 is Met(-1)-IGF-I of low purity (44%), and that the assigned protein content is higher than the value determined by quantitative amino acid analysis. Thus, assays that are calibrated against WHO IRR 87/518 will report total IGF-I concentrations in excess of actual values. We believe that calibration against WHO IRR 87/518 is the cause of the systematic discrepancy between the Genentech IGF-I assay normal range and most other normal ranges, and that much of the plasma IGF-I concentration data in the literature are of questionable accuracy.     

高纯金属钛中微量氢的分析方法研究

采用高频感应热导法测定高纯金属钛中氢的质量分数.着重讨论了感应电流、坩埚类型、助熔剂、空白值、最小分析时间及样品质量对测定结果的影响,制定出了高纯金属钛中氢的分析方法.结果表明,该方法测定高纯金属钛试样中氢质量分数的相对标准偏差(RSD)为6.0%,加标回收率为98%～106%.利用综合测定程序,探讨了金属钛中氢分量的测试条件,并通过分峰实验计算了各氢分量的值.     

Sensitive densitometry for the determination of platelet-activating factor and other phospholipids in human tears

A sensitive TLC method is reported for the determination of the platelet-activating factor (PAF) and other phospholipids in human tears. Tear samples absorbed on filter-paper were subjected to diatomite column extraction with chloroform-methanol. The eluent containing phospholipids was spotted on a silica gel plate. After removing lipids other than phospholipids by pre-development with hexane-diethyl ether (4 + 1 v/v), individual phospholipid separation was carried out by development with chloroform-methanol-water (65 + 35 + 7 v/v). Phospholipase C and then alkaline phosphatase solutions were sprayed on the TLC plate at 45 degrees C to liberate phosphate from each phospholipid. By spray application of a mixture of ammonium molybdate and Malachite Green, the liberated phosphates appeared as blue-green spots of molybdophosphate-Malachite Green aggregate on a yellow-brown background. The absorbance of each spot on the plate was measured at 620 nm with a densitometer. The peak area was found to be linearly related to PAF content in the range 2-100 pmol per spot, the RSD being 2% (n = 7). Approximately 86% of standard PAF added to tears was recovered. By this method, lysophosphatidylcholine (lysoPC), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in human tears could also be detected with high sensitivity. Each phospholipid in healthy human tears showed a nearly constant content; PAF, lysoPC, PC and PE were present at levels of 26.2, 42.3, 10.0 and 19.7%, respectively.     

Simultaneous quantitative gas-chromatographic analysis of ethosuximide, phenobarbitone, primidone and diphenylhydantoin

1 Therapeutic serum concentrations of ethosuximide, phenobarbitone, primidone, and dipheylhydantoin were assayed from 1 ml of human serum. The extraction procedure was common to all four drugs and three internal standards. 2 Subsequent isothermal gas chromatographic analysis of serum extracts produced well resolved peaks for the underivatized quantitation of ethosuximide and phenobarbitone. Primidone and diphenylhydantoin were determined as methylated derivatives. 3 Mean coefficients of variation for the assay of each drug were less 7% on a newly packed and conditioned column and less than 10% after the technique had been in continuous use for 3 months. 4 The advantage of quantitation relative to peak area ratios rather than peak height ratios was minimal for the determination of ethosuximide, primidone and diphenylhydantoin but appeared significant for the assay of phenobarbitone.