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PubMed2006

Quantitative determination of 20-hydroxyecdysone in methanolic extract of twigs from Vitex polygama Cham.

20-Hydroxyecdysone (20E) is effective in stimulating protein synthesis, therefore, it has been largely used as anabolic agent in several commercial formulas. Phytochemical study of methanolic extract of twigs from Vitex polygama, used in traditional Brazilian medicine as emenagogue, yielded a large quantity of 20E. This finding led us to developing and validating a simple and reliable method to determine 20E in the surveyed extract. Chromatographic separation of 20E was achieved on a phenyl-hexyl-based column using reversed elution mode. Extract was cleaned-up by solid phase extraction employing C(18) cartridge, and an absolute recovery of 97% was acquired. External standard and standard addition calibration graphs were obtained and good linearity was accomplished (r>0.999 for both curves). The limit of quantification and detection were determined. The results for accuracy fell within the -5 to +7% range.

Chromatography, High Pressure Liquid↗

PubMed2006

Development and validation of a sensitive liquid chromatography/electrospray tandem mass spectrometry assay for the quantification of olanzapine in human plasma.

A simple, sensitive and rapid liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was developed and validated for the quantification of olanzapine, atypical antipsychotic drug, in human plasma using loratadine as internal standard (IS). Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse phase C18 column and analyzed by MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 313/256 for olanzapine and m/z 383/337 for the IS. The assay exhibited a linear dynamic range of 0.1-30 ng/mL for olanzapine in human plasma. The lower limit of quantification was 100 pg/mL with a relative standard deviation of less than 10%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The average absolute recovery of olanzapine from spiked plasma samples was 85.5+/-1.9%. A run time of 2.0 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.

Antipsychotic Agents↗

PubMed2006

Liquid chromatographic tandem mass spectrometry method for the quantification of miglitol in human plasma.

A rapid, sensitive and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of miglitol (CAS 72432-03-2), an alpha-glucosidase inhibitor, in human plasma using gabapentin (CAS 60142-96-3) as internal standard (IS). Following protein precipitation, the analytes were separated using an isocratic mobile phase on a reversed phase phenyl column and analyzed by MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 208/146 for miglitol and m/z 172/154 for the IS. The assay exhibited a linear dynamic range of 100-6000 ng/mL for miglitol in human plasma. The lower limit of quantification was 100 ng/mL with a relative standard deviation of less than 5 %. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. The average absolute recoveries of miglitol and the IS from spiked plasma samples were 40.5 +/- 2.7 and 47.1 +/- 2.9 %, respectively. A run time of 2.5 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies. The miglitol plasma concentration profile could be obtained for pharmacokinetic study. The observed maximum plasma concentration (Cmax) of miglitol (100 mg oral dose) is 1740 ng/mL, time to observed maximum plasma concentration (tmax) is 3.5 h and elimination half-life (t(1/2)) is 2.5 h.

1-Deoxynojirimycin↗

PubMed2001

Assessment of skeletal muscle ventricle tissue blood flow using positron emission tomography.

In this pilot study, we assessed the feasibility of using positron emission tomography (PET) imaging for in vivo measurement of skeletal muscle ventricle (SMV) tissue blood flow. In 4 dogs, with SMVs prepared from their latissimus dorsi muscle, we quantified SMV tissue blood flow by PET and related it to the tissue flow measured by radiolabeled microspheres under similar physiologic conditions. The tissue blood flow was estimated in SMVs wrapped around a mandrel (not in circulation) at rest and during SMV stimulation (30 and 90 contraction-cycles/min). SMV tissue perfusion was heterogeneous, especially during SMV contraction. Furthermore, there was a linear relationship between SMV tissue flows estimated by PET and those measured by microspheres. We conclude that in vivo imaging of SMV is feasible by PET. Quantification of SMV tissue blood flow by PET has promise as a means of assessing changes in blood flow, but further technical progress needs to be made before absolute flows can be reliably measured.

Animals↗

PubMed1994

Influence of apolipoprotein(a) phenotype on lipoprotein(a) quantification: evaluation of three methods.

Three commercially available assays (an enzyme-linked immunosorbent assay ELISA, an immunoradiometric assay, IRMA, and a nephelometric assay) for the determination of lipoprotein(a) [Lp(a)] were compared with respect to the dependency of these assays on the various apolipoprotein(a) [apo(a)] isoforms. Although there was a strong correlation between the three methods, a significant difference between the absolute values (mg/L) was observed (p < 0.001). Using purified Lp(a) preparations, we showed that the ELISA assay quantifies the Lp(a) concentration on a molar basis, independently of the apo(a) isoform size. The IRMA and the nephelometric assay however are apo(a) isoform size dependent and overestimate the Lp(a) concentration of large apo(a) isoforms whereas the amount of small apo(a) isoforms is underestimated. In general, the isoform dependency of the Lp(a) quantification is of limited clinical relevance. In this study, inconsistent risk assignments are made in approximately 3% of the cases, when the Lp(a) concentrations obtained with the apo(a) isoform dependent assays are compared with the isoform independent ELISA.

Apolipoproteins A↗

PubMed2007

Isoprostane measurement in plasma and urine by liquid chromatography-mass spectrometry with one-step sample preparation.

BACKGROUND: Isoprostane F(2alpha) (iPF(2alpha)-III) concentration in plasma and urine is widely accepted as a measure of oxidative stress. Gas chromatography-mass spectrometry (GC/MS) methods for measuring iPF(2alpha)-III involve several steps of sample preparation and are labor-intensive, and ELISA methods, although easier to use, are less reliable. Therefore we developed a simple and sensitive method involving 1-step sample cleanup and HPLC/MS quantification. METHODS: Samples of plasma or urine were enriched with a deuterated (iPF(2alpha)-III-D4) standard, treated with KOH to liberate the bound isoprostanes, then loaded onto an immunoaffinity column, and the bound isoprostane was eluted with 95% ethanol. The concentrated sample was injected onto a C-18 HPLC column, and the isoprostane was eluted with a gradient of acetonitrile in water and analyzed by electrospray negative ionization, selectively monitoring the ions 353.2 (iPF(2alpha)-III) and 357.2 (iPF(2alpha)-III-D4). The amount of isoprostane in the sample was calculated from the ratio of the intensities of the 2 ions. RESULTS: The described method has a detection limit of 0.5 ng/L, with a linear dynamic range of 1-5000 ng/L. The intra- and interassay imprecisions were 4.68% and 3.88%, respectively. The values obtained correlated strongly with the GC/MS procedure (r = 0.80), but the absolute values were approximately 4- to 5-fold lower, because the present method measures specifically 1 isomer of isoprostane, whereas the GC/MS method measures 4 isomers together. CONCLUSIONS: Because of its simplicity and lower limit of quantification, the present method provides a useful noninvasive tool for determining oxidative stress in patients.

Adult↗

PubMed2005

Quantitative NMR in synthetic and combinatorial chemistry.

The applications of quantitative NMR to synthetic organic chemistry are reviewed with taking into account both the small libraries (100-150 compounds) and the single, well-characterized substance. The precision and accuracy which are obtained with state of the art instrumentation--both around 1%--rival with other classical tools of quantitative analytics, and qNMR does not require a specific method setup or a standard of the same substance. This characteristic makes it the method of choice in an environment where many different molecules are investigated and reliable quantification is required. NMR may effectively replace other standard characterization tools, such as CHNS analysis, or even complex, multi-determination results as commonly required for the assessment of absolute purity or strength of a substance, when no specific standard is available. Finally, because of the high precision and intrinsic accuracy, quantitative NMR appears the ideal reference method for the validation of other, more rapid, generic techniques for quantitative analysis.

Combinatorial Chemistry Techniques↗

PubMed1997

Hypoxia marker labeling in tumor biopsies: quantification of labeling variation and criteria for biopsy sectioning.

BACKGROUND AND PURPOSE: The error associated with using biopsy-based methods for assessing parameters reflective of the tumor microenvironment depends on the variability in distribution of the parameter throughout the tumor and the biopsy sample. Some attention has been given to intratumoral distribution of parameters, but little attention has been given to their intrabiopsy distribution. We evaluated the intrabiopsy distribution of CCI-103F, a 2-nitroimidazole hypoxia marker. MATERIALS AND METHODS: The hypoxia marker CCI-103F was studied in dogs bearing spontaneous solid tumors. Two biopsies were taken from each of seven tumors, for a total of 14 biopsies. Biopsies were serially sectioned and four to six contiguous slides from each 100-150 microm of the biopsy were used to formulate the best estimate of CCI-103F labeled area throughout the biopsy sample. One, two or four slides were then randomly selected from each biopsy and the labeled area, based on this limited sample, was compared to the estimate obtained from counting all available slides. Random sampling of slides was repeated 1000 times for each biopsy sample. RESULTS: CCI-103F labeling variance throughout the biopsy decreased as the estimated overall labeled area in the biopsy decreased. The error associated with estimating the overall labeled area in a biopsy from a randomly selected subset of slides decreased as the number of slides increased, and as the overall labeled area in the biopsy decreased. No minimally labeled biopsy was classified as unlabeled based on limited sampling. CONCLUSION: With regard to CCI-103F labeling, quantification of the labeled area in four randomly selected slides from a biopsy can provide, in most biopsies, an estimate of the labeled area in the biopsy within an absolute range of +/-0.05.

Animals↗

PubMed1991

A high-performance liquid chromatographic assay for the enantiomers of bevantolol in human plasma.

A method was developed and validated for the simultaneous analysis of (+)- and (-)-bevantolol in human plasma. The assay involves plasma protein precipitation, derivatization of racemic bevantolol to its diastereomeric thioureas with 2,3,4,5-tetra-o-acetyl-alpha-D-glucopyranosyl isothiocyanate, and solid-phase extraction of the diastereomers from 0.5 ml human plasma. Chromatographic separation was accomplished under isocratic conditions using a reversed-phase C-18 analytical column and mobile phase consisting of equal parts of 75 mM dibasic ammonium phosphate buffer (adjusted to pH 3.5 with phosphoric acid) and acetonitrile, with a detection wavelength of 220 nm. The absolute peak-height method was employed for quantitation. Retention times for the diastereomers of (+)- and (-)-bevantolol were 7.4 and 6.4 min, respectively. The method is suitable for the quantification of the enantiomers over a concentration range of 40 to 800 ng/ml per enantiomer.

Adrenergic beta-Antagonists↗

PubMed1997

[Functional evaluation of intermittent claudication].

Despite new technologies and therapeutical developments in angiology, the functional evaluation of intermittent claudication still remains unsatisfying. The Fontaine-Classification from 1954, which is currently used as stage IIa and IIb, is not generally accepted. In the clinical routine, the estimate taken for decision strategy, even for the interventional and surgical procedures, is often that claimed by the patient in his case history. Due to lack of standardization the results of therapeutical studies are not comparable. Recommendations for treadmill exercise testing are confusing. Globally, both the constant-load-test at 3 km/h and 12% grade and the graded-exercise-test (3,2 km/h with an increase in grade of 3,5% every 3 minutes) show similar results. However, in patients with absolute claudication distance between 50-150 m, the constant-load-test is superior. The problem in measurement of the treadmill walking distance could probably be solved by the quantification of exercise capability and evaluation of physical capacity in patients with intermittent claudication in the same manner, as usual in cardiology and sports medicine.

Exercise Test↗

PubMed1989

Do pharmacological methods for the quantification of agonists work when the ternary complex mechanism operates?

It is well established that many receptors couple to G-proteins in order to subserve their pharmacological or physiological effects. In those systems it is possible that a ternary complex mechanism operates in which initiation of an effect depends on the concentration of agonist-receptor-G-protein complex formed. Such systems may be considered to obey a receptor-transducer model (Black & Leff, 1983, Proc. R. Soc. B220, 141). A theoretical analysis of this model is presented which seeks to determine how the operation of the ternary complex mechanism affects the quantification of agonists by conventional pharmacological methods. Previous analyses have concluded that pharmacological models may or may not accommodate the ternary complex mechanism depending upon the relationship between the relative concentrations of receptor and transducer units, [R0], and [T0] respectively. The present study extends these in two ways. It considers the impact of the ternary complex mechanism on agonist quantification under a more complete range of conditions relating [R0] and [T0], and it does so with regard to the analysis of partial agonists (by the comparative method) as well as of full agonists (by the method of receptor inactivation). The following predictions are made: (i) reliable estimates of affinity and efficacy can be made using the comparative method under the conditions [R0] much greater than [T0] and [R0] much less than [T0] whereas the inactivation method only works under the former condition; (ii) errors occur in the estimation of affinity and efficacy by both methods when [R0] = [T0] although better estimates are produced by the comparative method; (iii) when errors occur in the absolute estimation of affinity and efficacy, the orders of affinity and efficacy determined by the comparative method will generally be correct but this is not the case for the inactivation method; (iv) in general, the comparative method for agonist quantification appears to produce more reliable information for the purposes of receptor classification and medicinal chemistry than does the receptor inactivation method.

Chemical Phenomena↗

PubMed1995

Regional quantification of dopamine transporter mRNA in rat brain using a ribonuclease protection assay.

We describe the regional distribution of dopamine transporter (DAT) mRNA in selected regions of rat brain using a highly sensitive and specific nuclease protection assay. This method determines the absolute quantity of mRNA expressed in the brain regions surveyed. DAT mRNA level varied widely between brain regions, and was detected only in cell body regions of the major dopaminergic pathways. Highest expression was seen in substantia nigra/ventral tegmentum (SN/VTA). Lower but detectable expression of a protected mRNA of the expected size was observed within hypothalamus. Expression could not be detected by this method in other brain regions studied. Our results indicate that this method is sufficiently sensitive to allow study of mRNA expression in individual animals.

Amino Acid Isomerases↗

PubMed2006

In vivo radionuclide uptake quantification using a multi-pinhole SPECT system to predict renal function in small animals.

PURPOSE: In vivo quantification of radiopharmaceuticals has great potential as a tool in developing new drugs. We investigated the accuracy of in vivo quantification with multi-pinhole single-photon emission computed tomography (SPECT) in rats. METHODS: Fifteen male Lewis rats with different stages of renal dysfunction were injected with 50 MBq 99mTc-dimercaptosuccinic acid. Four to six hours after injection, SPECT of the kidneys was acquired with a new four-headed multi-pinhole collimator camera. Immediately after imaging the rats were sacrificed and the kidneys were counted in a gamma-counter to determine the absorbed activity. SPECT data were reconstructed iteratively and regions of interest (ROIs) were drawn manually. The absolute activity in the ROIs was determined. RESULTS: Uptake values ranging from 0.71% to 21.87% of the injected activity were measured. A very strong linear correlation was found between the determined activity in vivo and ex vivo (r2=0.946; slope m=1.059). CONCLUSION: Quantification in vivo using this multi-pinhole SPECT system is highly accurate.

Animals↗

PubMed2004

Automated, fast, and sensitive quantification of drugs in human plasma by LC/LC-MS: quantification of 6 protease inhibitors and 3 nonnucleoside transcriptase inhibitors.

An analytic assay based on automated sample preparation and liquid chromatography (LC) coupled with electrospray mass spectrometry (ESI-MS) was developed for the quantification of 6 protease inhibitors (PIs) and 3 nonnucleoside reverse transcriptase inhibitors (NNRTIs). The 6 PIs, amprenavir, indinavir, ritonavir, lopinavir, nelfinavir, and saquinavir, as well as the three NNRTIs, nevirapine, efavirenz, and delavirdine, require a succinct analysis technique for therapeutic drug monitoring in HIV/AIDS patients. After protein precipitation, samples were loaded on a C8, 10 x 4-mm extraction column, washed, and, after activation of the column-switching valve, backflushed onto the 30 x 2.1 mm C8 analytic column. [M+H] ions were detected in the selected ion mode. A nonlinear fit (y(-1) = a + b/x, all r2 > 0.999) for amprenavir, indinavir, ritonavir, lopinavir, nelfinavir, and saquinavir and a linear fit (y = ax + b, all r2 > 0.999) for nevirapine, efavirenz, and delavirdine led to best regression. Absolute recoveries were as follows: PIs > 81%; NNRTIs > 76%. Interday and intraday precision were <12.5% for the PIs and <11.7% for the NNRTIs. Interday and intraday accuracy were <12.2% for the PIs and <14.9% for the NNRTIs. Limits of quantification were 20, 40, 50, 40, 40, 20, and 100 microg/L for amprenavir, indinavir, ritonavir, lopinavir, nelfinavir, saquinavir, and the NNRTIs, respectively. The assay allows fast analysis of patient samples for therapeutic drug monitoring (TDM) and has successfully been used for TDM and pharmacokinetic drug-drug interactions studies.

Anti-HIV Agents↗

PubMed2005

Quantification of cerebral perfusion using arterial spin labeling: two-compartment models.

One of the advantages of arterial spin labeling (ASL) techniques over other techniques for measuring cerebral perfusion is that with ASL it is possible to achieve accurate quantification. This is particularly useful in the field of functional imaging, where accurate measurements of perfusion change can help untangle the complex physiological changes that occur following neuronal activation. However, the linearity of the perfusion estimate over a wide range of perfusion values may be more important than absolute values. For several years, single-compartment models have dominated the literature, and it has been assumed that the labeled water diffuses freely throughout the tissue voxel. However, recent work, as summarized in this review, has shown that this assumption is inaccurate and leads to an overestimation of perfusion at low perfusion rates, and an underestimation at high rates. The inclusion of restricted permeability of the capillary wall to water in a two-compartment model offers improved quantification.

Arteries↗

PubMed1992

Radioisotopic evaluation of renal transplants.

Owing to the noninvasive nature, ready availability, and efficacy, radionuclide studies remain widely utilized following renal transplantation for monitoring changes in the functional status and detection of detrimental complications of the grafted kidney. Whereas surgical complications, including vascular occlusion, urine extravasation, drainage obstruction, hematoma, or lymphocele formation, can often be detected effectively, specification of other underlying causes of deterioration of parenchymal function, including acute tubular necrosis (ATN), various types of rejection, and cyclosporine A nephrotoxicity (CyA-NT), frequently cannot be derived independently from the findings of a study without clinical correlation. Besides imaging, plotting of renogram or time/activity curves, numerous quantitative methods have been introduced to provide objective measurements of the blood flow, as well as to gauge the capability of concentration and excretion of the transplanted kidneys. However, the findings whether qualitative or quantitative all have overlapping zones. There is no abnormal image, graphic, or numeric index absolutely specific for any of the possible posttransplant renal parenchymal complications. The differentiation of such conditions may best be achieved through chronologic association of the sequential changes, with or without quantification, detected in serial studies with the clinical presentation and findings.

Humans↗

PubMed2006

Simplified method for determination of polycarbamate fungicide in water samples by liquid chromatography with tandem mass spectrometry following derivatization with dimethyl sulfate.

A simple and sensitive analytical method for the determination of polycarbamate in water samples was developed. In this method, polycarbamate was cleaved under alkaline conditions and derivatized with dimethyl sulfate to methyl dimethyldithiocarbamate (DMDC-methyl) and dimethyl ethylenebisdithiocarbamate (EBDC-dimethyl). After the solid-phase extraction of the resulting methyl derivatives, they were measured by liquid chromatography with tandem mass spectrometry (LC-MS/MS), based on reversed-phase separation and MS/MS detection with positive atmospheric pressure chemical ionization. The absolute recoveries (mean+/-SD) all through the procedure from polycarbamate to DMDC-methyl and EBDC-dimethyl were 62.6+/-4.3 and 73.5+/-5.9%, respectively. The limits of detection and quantification of polycarbamate in the water samples were 0.061 and 0.20 microg/L in the form of DMDC-methyl, and 0.032 and 0.11 microg/L in the form of EBDC-dimethyl, respectively. The method was validated at levels of 0.25, 1.0, and 5.0 microg/L in the tap water and river water samples, and accuracy was achieved in the range of 94-109%. The proposed method can be applied to the determination of trace amounts of polycarbamate in environmental water samples.

Chromatography, Liquid↗

PubMed1995

Are fine-needle breast aspirates representative of the underlying solid tumour? A comparison of receptor levels, ploidy and the influence of cytokeratin gates.

Fifty-three solid and 33 fine-needle aspirate (FNA) samples (20 paired) of human breast carcinomas were examined by flow cytometry. Experiments were conducted to assess whether FNA samples were phenotypically representative of the solid tumour. Quantification of oestrogen receptor (ER), epidermal growth factor receptor (EGFR), c-erbB-2 receptor levels and ploidy were examined on the total and cytokeratin-positive cell populations. The absolute number of molecules of cytokeratin per cell expressed on the FNA (n = 33) and solid tumour (n = 53) samples showed no significant difference, but, on a proportional basis, there was a significant difference between the two samples (P = 0.004), with lower expression exhibited by the FNAs. Examination of paired data showed no significant difference in the percentage of cytokeratin-positive cells (P = 0.51) or in the number of cytokeratin molecules expressed (P = 0.25). While the correlation for ER expression between paired tumour and FNA samples in the absence of cytokeratin gating was P = 0.06, r2 = 0.18, clear correlation was shown when a cytokeratin gate was used (P = 0.005, r2 = 0.4). Repeating this experiment for EGFR, it was found that no correlation was seen between FNA and solid tumour (P = 0.2, r2 = 0.14) in ungated populations, but use of the cytokeratin gate improved the correlation (P = 0.05, r2 = 0.3). A similar finding was seen with c-erbB-2 expression (P = 0.2, r2 = 0.1) without cytokeratin gating and when it was employed (P = 0.05, r2 = 0.4). Ploidy data showed concordance in 18/20 cases. Three cases of aneuploidy were missed by FNA, and this was because of an insufficient number of cells for analysis. The presented data suggest that FNAs are representative of solid tumours and may be useful for measuring receptor levels on clinical material when cytokeratin gating is used. However, observation by light microscopy is still necessary to confirm the presence of tumour cells in FNAs subjected to flow cytometry.

Biopsy, Needle↗