The Royal Canadian Mounted Police (RCMP) Forensic Laboratory Services (FLS) needed a method to confirm positive lysergic acid diethylamide (LSD) immunoassay screening results. As a result, an ultra performance liquid chromatography tandem mass spectrometry (UPLC¢â/MS/MS) method was validated for the confirmation and quantitation of LSD, iso-LSD, N-demethyl-LSD (nor-LSD), and 2-oxo-3-hydroxy-LSD (O-H-LSD). The method was validated in urine and whole blood, where linearity, accuracy, precision, sensitivity, stability, selectivity, recovery, matrix effects, and reproducibility were evaluated. <p>The method involved a liquid-liquid extraction (LLE) of the analytes and the deuterated internal standard from 1 mL of urine or whole blood with dichloromethane:isopropyl alcohol after being basified. The average recovery for all analytes was ¡Ã 62%, and the matrix effect was found to be insignificant. MS/MS analysis was conducted with a triple quadrupole mass spectrometer by positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode. The lowest limit of quantitation (LLOQ) was 20 pg/mL for LSD and iso-LSD, and 50 pg/mL for nor-LSD and O-H-LSD. The method was linear, accurate, precise, selective, and reproducible from 20 to 2000 pg/mL for LSD and iso-LSD, and from 50 to 2000 pg/mL for nor-LSD and O-H-LSD with an r2 ¡Ã 0.99. <p>The refrigerated and frozen long term stability was investigated for 90 days. LSD was stable at all temperatures for 90 days. Iso-LSD in blood was also stable at all temperatures for 90 days, but iso-LSD in urine showed an initial decrease followed by a gradual increase back to day 0 concentrations. Nor-LSD was stable at all temperatures up to day 14, with >43% decrease by day 30, with no additional decrease for the next 60 days. O-H-LSD in urine was stable at all temperatures for 90 days, but by day 90 O-H-LSD in whole blood stored refrigerated decreased in concentration by >37%. Additionally, a case sample that was stored at -50¡ÆC for ten years was found to still contain measurable amounts of each compound. <p>The method was applied to blind samples and a case that screened positive with immunoassay. Retention time, relative retention time, and ion ratios were used as identification parameters and found to correctly identify the analytes 100% of the time with no false positives. The case sample showed that the concentration of O-H-LSD was 4 times greater than LSD in urine. Furthermore, both the detection of O-H-LSD in a blood case sample, and LSD in a vitreous humor case sample were the first to be documented.

A reverse-phase high pressure liquid chromatographic method for the quantitative analysis of unchanged ascorbic acid in human urine is described. Selection of an appropriate mobile phase and discussion of some of the analysis problems are presented. Twenty-four hour ascorbate excretion profiles from two subjects were determined. Standard redox titration procedures were used to corroborate the chromatographic method. When compared to classical titration or colorimetric redox procedures, the new assay features straightforward sample preparation and improved sensitivity.Ball State UniversityMuncie, IN 47306

The Royal Canadian Mounted Police (RCMP) Forensic Laboratory Services (FLS) needed a method to confirm positive lysergic acid diethylamide (LSD) immunoassay screening results. As a result, an ultra performance liquid chromatography tandem mass spectrometry (UPLC¢â/MS/MS) method was validated for the confirmation and quantitation of LSD, iso-LSD, N-demethyl-LSD (nor-LSD), and 2-oxo-3-hydroxy-LSD (O-H-LSD). The method was validated in urine and whole blood, where linearity, accuracy, precision, sensitivity, stability, selectivity, recovery, matrix effects, and reproducibility were evaluated. <p>The method involved a liquid-liquid extraction (LLE) of the analytes and the deuterated internal standard from 1 mL of urine or whole blood with dichloromethane:isopropyl alcohol after being basified. The average recovery for all analytes was ¡Ã 62%, and the matrix effect was found to be insignificant. MS/MS analysis was conducted with a triple quadrupole mass spectrometer by positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode. The lowest limit of quantitation (LLOQ) was 20 pg/mL for LSD and iso-LSD, and 50 pg/mL for nor-LSD and O-H-LSD. The method was linear, accurate, precise, selective, and reproducible from 20 to 2000 pg/mL for LSD and iso-LSD, and from 50 to 2000 pg/mL for nor-LSD and O-H-LSD with an r2 ¡Ã 0.99. <p>The refrigerated and frozen long term stability was investigated for 90 days. LSD was stable at all temperatures for 90 days. Iso-LSD in blood was also stable at all temperatures for 90 days, but iso-LSD in urine showed an initial decrease followed by a gradual increase back to day 0 concentrations. Nor-LSD was stable at all temperatures up to day 14, with >43% decrease by day 30, with no additional decrease for the next 60 days. O-H-LSD in urine was stable at all temperatures for 90 days, but by day 90 O-H-LSD in whole blood stored refrigerated decreased in concentration by >37%. Additionally, a case sample that was stored at -50¡ÆC for ten years was found to still contain measurable amounts of each compound. <p>The method was applied to blind samples and a case that screened positive with immunoassay. Retention time, relative retention time, and ion ratios were used as identification parameters and found to correctly identify the analytes 100% of the time with no false positives. The case sample showed that the concentration of O-H-LSD was 4 times greater than LSD in urine. Furthermore, both the detection of O-H-LSD in a blood case sample, and LSD in a vitreous humor case sample were the first to be documented.

Reproduction de : Thèse d'exercice : Médecine vétérinaire : Toulouse 3 : 2007. / Titre provenant de l'écran titre. Bibliogr. p. 65-68.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 111-120).

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

This work is a quantitative description of the renal excretion and the post-renal modification of ureteral urine from native (unanesthetized, uninfused, and normal hydropenic) desert quail, Lophortyx gambelii. The technique used in this study establishes the glomerular filtration rate (GFR), urine flow rate, and urinary excretion of water, sodium, potassium, and uric acid for desert quail in a relatively undisturbed state and in steady-state balance with regard to intake and output of water, sodium, and potassium. In contrast, conventional methods of determining GFR in birds include the use of anesthesia, cloacal or ureteral canulation, and infusion of fluids to introduce filtration markers (e.g. inulin) and to cause a diuresis (e.g. by using mannitol). In the present study, native desert quail had a urine flow rate of about 40 g/kg.day compared to over 500 g/kg.day for desert quail previously studied using conventional methods. Also in the present study, GFR was about 1.6 ml/kg.min which is about 25% lower than previously reported (2.1 ml/kg.min) for desert quail studied with conventional techniques. Renal absorption of the filtered loads of water, sodium and potassium also was determined in the present study. The fractions of the filtered loads reabsorbed by the renal tubules were: for water 98%, for sodium 99.4%, and for potassium 42%. These findings illustrate that renal reabsorption of these filtered substances is less complete in birds than in mammals where, in man for example, about 99% of the water and 99.8% of the sodium are normally reabsorbed. In addition, this study evaluates the role of the cloaca and lower intestines in changing the composition of the ureteral urine. Ureteral urine is modified in the cloaca and lower intestines of the desert quail before being excreted with the final droppings. This modification results in reabsorption of about 70% of the water and sodium and about 80% of the potassium in the ureteral urine. Thus for the desert quail, post-renal reabsorption of water and sodium from ureteral urine produced by the kidneys increases the total amounts of the filtered loads reabsorbed to 99% for water and 99.7% for sodium, which are nearly the same as seen for man. It is concluded that post-renal reabsorption of water and sodium is an important aspect of fluid and electrolyte balance in native desert quail. About 65% of the uric acid present in the ureteral urine was found to be degraded during its passage into the lower intestines. This is particularly significant because trapping of sodium and potassium occurs within the uric acid precipitates which form in bird urine. It was determined that about 20% of the sodium and 33% of the potassium in the ureteral urine are trapped within uric acid precipitates. Degradation of uric acid may increase the reabsorbable pools of these cations and facilitate their reabsorption by the tissues of the lower intestines. Since the intestinal ceca of birds contain large populations of uric acid-decomposing bacteria, and because other studies have suggested large amounts of water are reabsorbed in the ceca of birds, the role of the ceca in post-renal modification of urine was evaluated. The results are not conclusive. Cecaectomized (Cx) birds showed only a transitory increase in water loss when compared to sham operated (Sh) birds. No difference in uric acid excretion was seen between Cx or Sh birds. Thus, no obligatory role for the ceca in post-renal reabsorption of water and electrolytes, or in degradation of uric acid, was evident.

No description available.

Background: The most commonly employed screening method for proteinuria is a semi- quantitative dipstick urinalysis, but it has been shown to be inaccurate in pregnancy. New developments in the assessment of proteinuria have included the use of urinary albumin measurements. The Clinitek Microalbumin Reagent Strip (Bayer Healthcare LLC, USA) is a semi-quantitative dipstick test. It is used to measure the spot urinary microalbumin to creatinine ratio that is read using the Clinitek 50 portable urine chemistry analyzer. Aims We embarked on a pilot study to validate the Clinitek 50 system by determining the accuracy of spot urinary microalbumin to creatinine ratio dipsticks and conventional visual dipsticks (Makromed) compared to the laboratory urinary microalbumin to creatinine ratio quantification to detect significant proteinuria in normotensive and hypertensive antenatal attendees. The accuracy of spot urinary microalbumin to creatinine ratio dipsticks and conventional visual dipsticks were then compared to a 24 hour urinary protein (gold standard) to detect significant proteinuria in hypertensive disorders of pregnancy. We then determined the role of proteinuria as assessed by the diagnostic accuracy of both the 24 hour urinary protein (gold standard) and the spot urinary microalbumin to creatinine ratio dipstick, in pregnancy outcomes of these participants. Methods This was a prospective study conducted at hospitals serving the Durban Metropolitan region in South Africa. To validate the urinary microalbumin to creatinine ratio dipstick, fifteen normotensive healthy pregnant women and 11 women with new onset hypertension in pregnancy were recruited .Each women had a spot midstream urine, which was assessed for proteinuria using a semi-quantitative visual dipstick (Makromed) and analysed using the semi-quantitative urinary microalbumin to creatinine ratio dipsticks (Clinitek® Microalbumin) read on the Clinitek® 50 urine chemistry analyser. A result of 1 + on visual dipsticks and a spot urinary microalbumin to creatinine ratio UAC of > 300mg/g (33.9mg/mmol) was considered as positive for significant proteinuria. The results were compared to the laboratory quantitative measurement of the urinary microalbumin to creatinine ratio. The study group comprised 163 women presenting with newly diagnosed hypertension during pregnancy after 20 weeks of gestation, being recruited from antenatal clinics. Each participant had a spot urine sample that was tested by trained midwives for proteinuria using a semi-quantitative visual dipstick (Makromed). Participants were admitted to the ward where a spot midstream urine sample was collected and analysed using the semi-quantitative urinary microalbumin to creatinine ratio dipsticks. A 24 hour quantitative urinary protein analysis was completed. The results of the urinary microalbumin to creatinine ratio dipsticks and conventional visual dipsticks were compared to the 24 hour urinary protein (gold standard) to detect significant proteinuria. A urinary microalbumin to creatinine ratio of < 300mg/g (nil and trace on visual urine dipsticks) was considered to be a negative result. A urinary microalbumin to creatinine ratio 300 mg/g (1+ to 4+ on visual urine dipsticks) was considered to be a positive result. Urinary protein 0.3 g/24 hours was considered significant proteinuria. The outcomes of pregnancy in 2 sub-categories viz. those with and without significant proteinuria were compared using the 24 hr urinary protein measurement. A secondary analysis of outcomes of pregnancy was performed by subcategorizing the participants according to the diagnostic accuracy of the urinary microalbumin to creatinine ratio dipsticks. In the 26 patients enrolled in the initial study , the visual dipstick had a sensitivity of 25% ( 95% CI [0.04-0.64] ) and specificity of 89% ( 95% CI [0.64 -0.98]).The urinary microalbumin to creatinine ratio dipsticks had a sensitivity of 88% ( 95% CI [0.47-0.99]), specificity of 89% (95% CI [0.64-0.98]), negative predictive value (NPV) of 94% (95% CI [0.69-1.00]) and positive predictive value (PPV) of 78% (95% CI [0.40-0.96]). In the 163 patients subsequently enrolled the visual dipstick had a sensitivity of 51 % ( 95% CI [0.41-0.61]) and specificity of 91% (95% CI [0.81-0.96]) .The PPV and NPV was 89 %( 95% CI [0.77-0.95]) and 58% (95% CI [0.48-0.67]) respectively. The urinary microalbumin to creatinine ratio dipsticks had a sensitivity of 63% (95% CI [0.52-0.72]) and specificity of 81 % (95% CI [0.70-0.89]). The PPV was 82% (95% CI [0.71-0.90]) and NPV was 62% (95% CI [0.51-0.71]). Our results show that in hypertensive pregnant women, significant proteinuria determined by the quantitative 24 hour urinary protein is associated with delivery at an earlier gestational age, increased induction of labour and lower birthweights compared to the non-proteinuric hypertensives (gestational hypertension). There is also a trend towards an increased maternal morbidity and perinatal mortality. When the groups were classified into pre-eclampsia and gestational hypertension using the diagnostic accuracy of the urinary microalbumin to creatinine ratio dipsticks, there were no differences in the clinical outcomes between the false negatives and true negatives except a trend towards a higher caesarean section rate in the false negatives. Conclusion The urinary microalbumin to creatinine ratio dipstick read on the Clinitek 50 system provides a semi – quantitative result of the urinary microalbumin to creatinine ratio that has good sensitivity and specificity. Furthermore, the urinary microalbumin to creatinine ratio dipstick has a good negative predictive value and a result of < 300mg/g rules out significant proteinuria and avoids unnecessary investigations in pregnancy. Both the visual dipstick (Makromed) and the urinary microalbumin to creatinine ratio dipstick read on the Clinitek 50 system are not accurate when compared to the total 24 hour urinary protein. Differences between the urinary microalbumin to creatinine ratio and 24 hour total urinary protein may be due to the variation in the albumin fraction of the total urinary protein of pre-eclampsia, technical problems with imprecision of the assay technique and clinical causes of false positives and negatives. The improved sensitivity of the automated urinary microalbumin to creatinine ratio dipstick over the visual dipstick suggests it may be a suitable substitute for the visual dipstick in clinical practice Hypertension in pregnancy associated with significant proteinuria is associated with greater adverse maternal and fetal outcome. Outcome of pregnancy is similar when a classification of gestational hypertension is made based either on the 24 hour urinary protein or the urinary microalbumin to creatinine ratio dipstick read on the Clinitek 50 system. The urinary microalbumin to creatinine ratio dipstick is a good screening test to rule out significant proteinuria. It has the potential to improve accuracy of screening for proteinuria and enhancing safety by preventing incorrect diagnosis and unnecessary investigation. Further research is required to determine its full impact and cost effectiveness in the clinical setting. / Thesis (M.Med.)-University of KwaZulu-Natal, 2008.