Arteriovenous carboxyhemoglobin difference in critical illness: fiction or fact?

It is still unclear whether the paradoxical arteriovenous carboxyhemoglobin (COHb) difference found in critical illness is due to increased COHb production by the lung, or whether this gradient is caused by technical artifacts using spectrophotometry. In healthy and matched endotoxemic sheep, blood gases were analyzed with a standard ABL 625 and the updated version, an ABL 725. The latter one was accurately calibrated for COHb wavelengths (SAT 100) to eliminate the FCOHb dependency on oxygen tension. All endotoxemic sheep exhibited a hypotensive-hyperdynamic circulation and a pulmonary hypertension. Interestingly, arteriovenous COHb difference occurred in both healthy and endotoxemic sheep (P<0.001 each). Arterial and central venous COHb concentrations determined with the ABL 625 were significantly lower than those measured with the ABL 725 (P<0.001 each). We conclude that (a) arteriovenous COHb difference per se does not reflect critical illness and (b) measurements with an ABL 625 underestimate COHb concentrations.     

Should we measure serum or plasma lead concentrations?

ProjectSerum samples may not be appropriate to assess lead (Pb) concentrations because they may contain artificially higher Pb concentrations compared with those measured in plasma samples. Here, we compared Pb concentrations in serum versus heparin plasma separated from blood collected with or without vacuum. We have also examined the effects of sample standing time on Pb concentrations measured in serum, heparin plasma, and EDTA plasma.ProcedureWe studied plasma and serum samples from twelve healthy subjects. Blood samples were collected via venous drainage phlebotomy with and without vacuum into trace metal free tubes containing no anticoagulants (serum), or lithium heparin, or EDTA (to obtain plasma). Variable sample standing times (0, 5, and 30 min) prior to centrifugation were allowed. Plasma and serum Pb and iron concentrations were determined by inductively coupled plasma mass spectrometry. Plasma and serum cell-free hemoglobin concentrations were measured.ResultsPb concentrations in serum and in heparin plasma from blood samples collected with or without vacuum were similar and not associated with significant changes in iron or hemoglobin concentrations. The sample standing time (up to 30 min) did not affect Pb concentrations in serum or in heparin plasma, which were approximately 50% lower than those found in EDTA plasma.ConclusionsSerum or heparin plasma separated from blood samples collected via venous phlebotomy with or without vacuum are appropriate medium to assess Pb concentrations, independently of the sample standing time.     

Evaluation of viscoelasticity measurements of human blood

The dependence on hematocrit of whole blood viscoelasticity must be considered in order to compare pathological blood samples to normal ones. If one wants to calculate the measured values to a standard hematocrit value, the hematocrit dependence for the pathological sample must be available. As the latter however is unknown, the same dependence is assumed for both normal and pathological blood samples. To prove the validity of this assumption, hematocrit dependence of random blood samples from different diseases (cerebral and coronary vascular and myocardial disorders) were investigated. A statistical analysis showed the assumption as invalid. Therefore, it will be recommended to evaluate pathological blood samples at the measured hematocrit.     

UNDERSTANDING AND INTERPRETING HEMATOCRIT MEASUREMENTS IN PINNIPEDS

Hematocrit data are relatively easily obtained from blood samples of pinnipeds but differences in methodology and variable physiological conditions of the subjects can significantly alter their values. This two-fold problem makes comparative data and modeling efforts difficult. To quantify the difficulty of obtaining accurate and representative hematocrit values in pinnipeds, hematocrit was measured by both microcentrifugation and Coulter counter methods in a range of pinnipeds under a variety of physiological and handling conditions. The data show that the Coulter counter hematocrit values were 4%-15% higher than those measured by microcentrifugation. In addition, blood samples from restrained animals showed consistently elevated hematocrit values relative to resting subjects. A significant difference was also found between hematocrit values from pups and adults. Finally, hematocrit was shown to decline over the course of isofluorane anesthesia. Taken together, these results suggest that laboratory methodology, developmental state, and animal handling techniques can significantly alter hematocrit values in pinnipeds. Thus, modeling efforts that require representative hematocrit values, such as calculations of total blood oxygen stores, can be markedly impacted by variations in hematocrit measurement techniques and sampling regimes.     

Regional lung hematocrit in humans using positron emission tomography

Regional lung hematocrit ratio (R) was measured in five normal subjects and five patients (2 with pneumonia, 2 with nephrotic syndrome with anemia, and 1 with pancreatitis) using positron emission tomography, a red cell marker 11CO, and a plasma marker [methyl-11C]albumin). The measurements were made in a transaxial thoracic section at midheart level with the subject in supine posture and with a spatial resolution of 1.7 cm. The normal regional hematocrit ratio (means +/- SE) calculated for the lung was 0.90 +/- 0.014, 0.94 +/- 0.023 for the thoracic wall, and 1.00 +/- 0.003 for the heart chambers. The regional lung hematocrit ratio in the patients ranged between 0.81 and 0.86. No correlation was found among the regional lung hematocrit ratio and regional blood volume, lung extravascular density, and the peripheral hematocrit (obtained from venous blood samples). To the extent that 70% of the pulmonary blood in the field of view is in larger vessels with normal hematocrit, the hematocrit in the capillary bed is approximately two-thirds that of the peripheral venous value. Blood volume measurements on the basis of single vascular tracers need to take account of these results.     

Iron status in runners of various running specialties.

The blood iron status of 44 male runners of various running specialties (18 sprinters, 13 middle- and 13 long-distance runners) is evaluated by measuring serum ferritin (SF), serum iron (Si), hemoglobin concentration (Hb), hematocrit (Ht), red blood cells content (RBC) and haptoglobin concentration (Hp). The results of these analyses (except Hp) are compared to those obtained in sedentary male subjects (control group) of the same mean age. Mean SF, SI, Hb and Ht measured in athletes are significantly lower than in control group. The remarkably low Hp values obtained in athletes suggests the occurrence of hemolysis. Using unpaired t test, it appears that the blood iron status of these runners does not depend on their running specialty.     

Determination of gatifloxacin in human serum and urine by high-performance liquid chromatography with ultraviolet detection

A simple and sensitive HPLC method for the determination of gatifloxacin concentrations in human serum and urine was developed and validated. Serum proteins were removed by ultrafiltration through a filtering device after adding a displacing agent. Urine samples were diluted with mobile phase prior to injection. Separation was achieved with a C18 reverse-phase column and gatifloxacin concentrations were determined using ultraviolet detection. The quantitation limits of the assay were 100 ng/ml in serum and 1.0 microg/ml in urine. The assay method was successfully applied to a pharmacokinetic study of gatifloxacin in healthy volunteers.     

Rapid determination of indomethacin and salicylic acid in serum by means of reversed-phase liquid chromatography

A method for the quantitative analysis of indomethacin and salicylic acid in blood serum and urine by high-performance liquid chromatography is described. A C₁₈-bonded silica was used as the stationary phase and mixtures of ethanol, n-butanol and aqueous buffer as the mobile phase. Before injection the serum is deproteinized and extracted in one step.The recovery of the extraction was found to be 88% and 77% for indomethacin and salicylic acid, respectively. The relative standard deviations of the analysis for 0.5 μg indomethacin and 5 μg salicylic acid per ml serum were 3.6% and 3.2%, respectively. The detection limits for indomethacin and salicylic acid were 2 ng. This corresponds for both substances to 0.1 μg/ml serum for an injection volume of 100 μl.The method enables simultaneous determination of possibly formed metabolites. A number of concurrently administered drugs do not interfere with the analysis. The interactive effects of co-medication of indomethacin and salicylic acid on the serum concentration of indomethacin is demonstrated by measuring the pharmacokinetic curves.     

Tobacco smoking causes secondary polycythemia and a mild leukocytosis among heavy smokers in Taif City in Saudi Arabia

Tobacco smoking is a common risk factor of cardiovascular diseases, cancers and heart health problems. In Taif, the number of secondary polycythemia patients is increasing dramatically and most of those patients are heavy smokers. Therefore, this study is an attempt to understand the pathophysiological mechanism behind that problem. Whole blood and serum samples were collected from forty healthy people and forty tobacco smokers, voluntary for this study. Complete blood counts revealed a significant increase in the red blood cell count, hemoglobin concentrations, hematocrit and neutrophils with some elevations in total white blood cells, lymphocytes and monocytes. Moreover, serum analysis of both erythropoietin and interleukin-7 showed a significant reduction in their levels among smokers which were about 35% and 65% respectively. Gene expression study showed a significant upregulation of RAG-1, RAG-2 and EPOR-1 genes caused by tobacco smoking. In conclusion, data presented in the current study suggest that tobacco smoking might cause alveolar tissue inflammation and vascular injury causing an immune response that elevates the white blood cells count. Another suggestion is that tobacco smoking defects the pulmonary gaseous exchange mechanism leading to the secondary polycythemia indicated by the increase in red blood cell count, hemoglobin levels, hematocrit and by the low serum erythropoietin levels.     

Determination of mercury in human serum and packed blood cells by neutron activation analysis

A method is described for the determination of mercury in human blood serum and packed blood cells employing neutron activation analysis. Great attention was devoted to the collection and manipulation of the samples. The accuracy and precision of the method were tested by analyzing biological reference materials and by comparing the concentrations measured in a number of serum samples to those obtained by another, independent technique (cold vapor atomic absorption spectrometry) in the same samples. The article reports the levels measured in blood serum and packed blood cells samples from 15 adult volunteers, as well as the figures determined in a “second-generation” biological reference material (freeze-dried human serum), prepared and conditioned at the University of Ghent.     

Dexamethasone quantification in dried blood spot samples using LC-MS: The potential for application to neonatal pharmacokinetic studies

A high-performance liquid chromatography (LC-MS) method has been developed and validated for the determination of dexamethasone in dried blood spot (DBS) samples. For the preparation of DBS samples whole blood spiked with analyte was used to produce 30μl blood spots on specimen collection cards. An 8mm disc was cut from the DBS sample and extracted using a combination of methanol: water (70:30, v/v) containing the internal standard, triamcinolone acetonide. Extracts were centrifuged and chromatographic separation was achieved using a Zorbax Eclipse Plus C18 column using gradient elution with a mobile phase of acetonitrile and water with formic acid at a flow rate of 0.2ml/min. LC-MS detection was conducted with single ion monitoring using target ions at m/z 393.1 for dexamethasone and 435.1 for the internal standard. The developed method was linear within the tested calibration range of 15-800ng/ml. The overall extraction recovery of dexamethasone from DBS samples was 99.3% (94.3-105.7%). The accuracy (relative error) and precision (coefficient of variation) values were within the pre-defined limits of ≤15% at all concentrations. Factors with potential to affect drug quantification measurements such as blood haematocrit, the volume of blood applied onto the collection card and spotting device were investigated. Although a haematocrit related effect was apparent, the assay accuracy and precision values remained within the 15% variability limit with fluctuations in haematocrit of ±5%. Variations in the volume of blood spotted did not appear to affect the performance of the developed assay. Similar observations were made regarding the spotting device used. The methodology has been applied to determine levels of dexamethasone in DBS samples collected from premature neonates. The measured concentrations were successfully evaluated using a simple 1-compartment pharmacokinetic model. Requiring only a microvolume (30μl) blood sample for analysis, the developed assay is particularly suited to pharmacokinetic studies involving paediatric populations.     

Free‐moving artificial eggs containing temperature loggers reveal remarkable within‐clutch variance in incubation temperature

Incubation is a crucial aspect of avian parental care and measuring incubation temperature in the wild can improve our understanding of life history tradeoffs and inform conservation efforts. However, there are challenges associated with measuring the temperature of eggs in natural nests. Most studies to date have measured incubation temperature by using a single, stationary logger in each nest. However, real eggs are rotated and moved throughout the nest by the parent during the incubation period, and thus, a stationary logger may not accurately represent the temperature experienced by individual eggs within the entire clutch. We recorded incubation temperature in nests by using multiple, mobile artificial egg temperature loggers. We installed six mobile loggers and one stationary logger in wood duck Aix sponsa nests to compare the two logger types in the field. We found that at a given ambient temperature, mobile loggers recorded lower average and more variable temperatures than stationary loggers. Further, temperatures recorded by stationary loggers showed no relationship with clutch size, while mobile loggers captured temperatures that were lower and more variable as clutch size increased. Also, the multiple mobile loggers revealed that eggs within a nest experienced a substantial range of temperatures throughout the incubation period. We discuss potential limitations of this method, but believe that it is a promising way to collect biologically‐relevant incubation temperature data and provides an opportunity to advance our understanding of incubation temperature as a parental effect.     

Validation of a Novel Collection Device for Non-Invasive Urine Sampling from Free-Ranging Animals

Recent advances in non-invasively collected samples have opened up new and exciting opportunities for wildlife research. Different types of samples, however, involve different limitations and certain physiological markers (e.g., C-peptide, oxytocin) can only be reliably measured from urine. Common collection methods for urine to date work best for arboreal animals and large volumes of urine. Sufficient recovery of urine is thus still difficult for wildlife biologists, particularly for terrestrial and small bodied animals. We tested three collection devices (two commercially available saliva swabs, Salivette synthetic and cotton, and cotton First aid swabs) against a control to permit the collection of small volumes of urine from the ground. We collected urine samples from captive and wild macaques, and humans, measured volume recovery, and analyzed concentrates of selected physiological markers (creatinine, C-peptide, and neopterin). The Salivette synthetic device was superior to the two alternative devices. Concentrations of creatinine, absolute C-peptide, C-peptide per creatinine, absolute neopterin, and neopterin per creatinine measured in samples collected with this device did not differ significantly from the control and were also strongly correlated to it. Fluid recovery was also best for this device. The least suitable device is the First aid collection device; we found that while absolute C-peptide and C-peptide per creatinine concentrations did not differ significantly from the control, creatinine concentrations were significantly lower than the control. In addition, these concentrations were either not or weakly correlated to the control. The Salivette cotton device provided intermediate results, although these concentrations were strongly correlated to the control. Salivette synthetic swabs seem to be useful devices for the collection of small amounts of urine from the ground destined for the assessment of physiological parameters. They thus provide new opportunities for field studies to incorporate physiological markers, particularly on smaller bodied and terrestrial animals and where urine collection is difficult.     

Thalidomide enantiomers: determination in biological samples by HPLC and vancomycin-CSP

Thalidomide is a racemate with potentially different pharmacokinetics and pharmacodynamics of the component (+)-(R)- and (-)-(S)-thalidomide enantiomers. As part of a project on the adjunctive effects of thalidomide and cytotoxic agents, a method for the chiral separation and quantitation of thalidomide was developed and validated. Thalidomide in relevant serum and tissue homogenate samples was stabilized by buffering with an equal volume of citrate-phosphate buffer (pH 2, 0.2M), and stored at -80 degrees C pending assay. The thalidomide enantiomers, extracted from the samples with diethyl ether, were well separated on a chiral HPLC column of vancomycin stationary phase and a mobile phase of 14% acetonitrile in 20 mM ammonium formate adjusted to pH 5.4; their concentrations were determined with phenacetin as internal standard at 220 nm detection. Over a thalidomide concentration range of 0.1-20 microg/ml, assay precision was 1-5% (CV) for both enantiomers, and calibration curves were linear with all correlation coefficients being >0.99. The estimated limit of quantification for both enantiomers was 0.05 microg/ml with 0.2-0.6 ml serum samples. Thalidomide in rat and human serum, acidified and stored as described above, was found to be chemically and chirally stable over 1 year. The method has been successfully applied to serum samples from human patients undergoing thalidomide treatment for mesothelioma, and to serum, blood and tissue samples from a laboratory rodent model using transplanted 9l gliosarcoma. Enantioselectivity in thalidomide pharmacokinetics has been found, thereby reinforcing the need for considering the relevance of chirality in thalidomide pharmacology.     

Capturing cancer cells using aptamer-immobilized square capillary channels

We report a simple square capillary-based cell affinity chromatography device that utilizes a coating of aptamers for selective capture of target cancer cells from a flowing suspension. The device consists of a square capillary with an inner diameter of roughly five cell diameters, connected via Teflon tubing to a syringe. Aptamers are immobilized on the inner surface of the capillary through biotin-avidin chemistry, the extent of which can be controlled by adjusting the aptamer concentration. Introduction of different cell types into separate devices, as well as mixtures of target and non-target cells, demonstrated that aptamer-target cells can be captured in significantly higher concentrations compared to non-target cells. Once optimized, 91.1 ± 3.5% capture efficiency of target leukemia cells was reported, as well as 97.2 ± 2.8% and 83.6 ± 5.8% for two different colon cancer cell lines. In addition, cells captured in the device were imaged, and the square capillary exhibited better optical properties than standard cylindrical capillaries, leading to the detection of leukemia cells in blood samples. Compared to current microfluidic cell affinity devices, this capture device requires no complicated design or fabrication steps. By providing a simple means of detecting and imaging cancer cells in the blood, this work has potential to directly assist clinicians in determining disease prognosis and measuring therapeutic response.     

Lactate determination in exercise testing using an electrochemical analyser: with or without blood lysis?

The practical use of lactate electrochemical analysers in exercise testing has not been adequately examined. Initial studies have reported differences in lactate concentration between that measured spectrophotometrically and that measured electrochemically. The study described here was undertaken to compare, using the statistical technique of Bland and Altman (1986), two widely available methods of measuring lactate using lysed and non-lysed blood samples and the lactate thresholds derived from the measured lactate values using a log-log transform technique. Thirteen normal, healthy young adults (11 male) undertook progressive exercise tests to exhaustion. Arterialised venous blood samples were taken each minute and the lactate concentration therein was measured both spectrophotometrically and electrochemically and either with or without lysis of the blood samples. The lactate concentrations measured in lysed blood using both methods (182 pairs) were in close agreement. The electrochemical values obtained using non-lysed blood were systematically lower than spectrophotometric values (206 pairs), the difference becoming progressively greater at higher lactate concentrations. Results for the lactate threshold comparisons are given as mean difference (limits of agreement with 95% probability). Lactate thresholds (12 pairs) derived from lysed blood lactate concentrations measured spectrophotometrically and electrochemically were not significantly different -30 (240) ml O2 x min(-1). Lactate thresholds (11 pairs) derived from lysed spectrophotometric and non-lysed electrochemical measurements were also not significantly different + 20 (250) ml O2 x min(-1). Thus, despite the difference in the measured lactate concentrations, the derived lactate thresholds are in agreement and, therefore, electrochemical analysers can be used for lactate threshold determination using the log-log transform technique without sample lysis.     

Measurement of cerebral blood volume using near-infrared spectroscopy and indocyanine green elimination.

Methods for measuring cerebral blood volume (CBV) have traditionally used radioisotopes. More recently, near-infrared spectroscopy (NIRS) has been used to measure CBV by using a technique involving O(2) desaturation of cerebral tissue, where the observed change in the concentration of oxygenated hemoglobin is a marker of the volume of blood contained within the brain. A new integration method employing NIRS is described by using indocyanine green (ICG) as the intravascular marker. After bolus injection, concentration-time integrals of cerebral tissue ICG concentration ([ICG](tissue)) measured by NIRS are compared with corresponding integrals of the cerebral blood ICG concentrations ([ICG](blood)) estimated by high-performance liquid chromatography of peripheral blood samples with allowance for cerebral-to-large-vessel hematocrit ratio. It is shown that CBV = integral [ICG]tissue/[ICG]blood. Measurements in 10 adult volunteers gave a mean value of 1.1 +/- 0.39 (SD) ml/100 g illuminated tissue. This result, although lower than previous NIRS estimations, is consistent with the long extracerebral path of light in the adult head. Scaling of results is required to take into account this component of the optical pathlength.     

Evaluation of a community-based automated blood pressure measuring device

Background

Automated devices are widely available in the community for people to measure their blood pressure. We assessed the accuracy and reproducibility of a brand of community-based automated device against the standard mercury sphygmomanometer.

Methods

Same-arm pairs of blood pressure readings were obtained with the Vita-Stat 90550 automated device, a sphygmomanometer and the Omron HEM-705CP automated device in random order on volunteers in 3 community pharmacies using a modified protocol for evaluating blood pressure devices. Comparison of readings between the Omron device and the sphygmomanometer served as a positive control of how well a laboratory-validated automated device could perform in the community. Both the Association for the Advancement of Medical Instrumentation (AAMI) and British Hypertension Society (BHS) criteria were used to assess the accuracy and reproducibility of readings.

Results

The mean blood pressure reading and standard error (SE) of the mean for the 108 volunteers (66 women and 42 men) was 133/77 (SE 2/1) mm Hg with the Vita-Stat device, 131/77 (SE 2/1) mm Hg with the Omron device and 129/76 (SE 2/1) mm Hg with the sphygmomanometer. The mean difference in readings was 4.4/1.0 (standard deviation [SD] 9.4/6.2) mm Hg between the Vita-Stat device and the sphygmomanometer and 1.6/0.6 (SD 9.3/6.4) mm Hg between the Omron device and the sphygmomanometer. Neither automated device met the AAMI accuracy criteria for the systolic readings. The BHS grades were C/A (systolic unacceptable/diastolic acceptable) for each automated device. According to the BHS analytical criterion, all devices achieved acceptable reproducibility grades.

Interpretation

Neither automated device met the AAMI or BHS criteria for accuracy while in use in the community, and neither performed as well in the community as in the laboratory.Measurement of blood pressure outside the office setting, using ambulatory monitors, home recorders or community-based devices has become popular among both physicians and patients. These devices may help to improve patients'' involvement in their care¹ and they may allay physicians'' concerns about a possible “white-coat syndrome.” However, incorrect readings could lead to a false sense of security or incorrect clinical decisions.The British Hypertension Society (BHS)² and the Association for the Advancement of Medical Instrumentation (AAMI)³ have developed laboratory protocols to evaluate automated blood pressure measuring devices. Many devices have failed to meet minimum standards for accuracy and reproducibility.⁴One community-based device, the Vita-Stat, has been available in various models since 1976, although none has performed uniformly well in community evaluations.^5,6,7,8,9 The newest model, the Vita-Stat 90550, available in about 3000 Canadian communities since 1990, provides 40 million readings yearly (Fred Sarkis, Spacelabs Medical: personal communication, 2000). Hence, we decided to evaluate the Vita-Stat 90550 against the mercury sphygmomanometer for accuracy and reproducibility in the community. To assess how well a laboratory-validated device could perform in the community, we compared the Omron HEM-705CP, which has met both the BHS and the AAMI criteria,¹⁰ against the mercury sphygmomanometer.     

Assay of inorganic sulfate in biologic fluids by nonsuppressed (single-column) ion chromatography

An assay using nonsuppressed (single-column) anion chromatography was developed to determine the concentration of inorganic sulfate in biologic fluids. A conventional HPLC system with an anion-exchange column and conductimetric detector interfaced with an automatic injector and integrator was used. The mobile phase for the chromatography of urine and serum samples is 4 mM potassium hydrogen phthalate, pH 4.5, and potassium iodide is used as the internal standard. For cerebrospinal fluid samples, the mobile phase is modified by addition of 10% of a 4 mM phthalic acid solution. Results of the HPLC assay were found to correlate well (r = 0.991 and 0.999) with those of two commonly used spectrophotometric methods for urine and serum inorganic sulfate determinations. However, the concentrations determined by ion chromatography were 2.5 to 10% lower, possibly due to less assay interference by other substances following chromatographic separation of sulfate. Anion chromatography using a single-column system is a convenient and relatively inexpensive method with sufficient sensitivity for the determination of inorganic sulfate concentrations in urine, serum, and cerebrospinal fluid.     

A dried blood spots technique based LC-MS/MS method for the analysis of posaconazole in human whole blood samples

A rugged and robust liquid chromatographic tandem mass spectrometric (LC-MS/MS) method utilizing dried blood spots (DBS) was developed and validated for the analysis of posaconazole in human whole blood. Posaconazole fortified blood samples were spotted (15 μL) onto Ahlstrom Alh-226 DBS cards and dried for at least 2h. Punched spots were then extracted by using a mixture of acetonitrile and water containing stable labeled internal standard (IS). Posaconazole and its IS were separated from endogenous matrix components on a Kinetex? C18 column under gradient conditions with a mobile phase A consisting of 0.1% formic acid and a mobile phase B consisting of 0.1% formic acid in acetonitrile/methanol (70/30, v/v). The analyte and IS were detected using a Sciex API 4000 triple quadrupole LC-MS/MS system equipped with a TurboIonSpray? source operated in the positive ion mode. The assay was linear over the concentration range of 5-5000 ng/mL. The inter-run accuracy and precision of the assay were -1.8% to 0.8% and 4.0% to 10.4%, respectively. Additional assessments unique to DBS were investigated including sample spot homogeneity, spot volume, and hematocrit. Blood spot homogeneity was maintained and accurate and precise quantitation results were obtained when using a blood spot volume of between 15 and 35 μL. Human blood samples with hematocrit values ranging between 25% and 41% gave acceptable quantitation results. The validation results indicate that the method is accurate, precise, sensitive, selective and reproducible.