Opiate detection in saliva and urine--a prospective comparison by gas chromatography-mass spectrometry.

UNLABELLED: There is an increasing interest in saliva as an alternative analytic body fluid. OBJECTIVE: This study sought to determine the correlation of opiates analyzed in saliva and corresponding urine. METHODS: A total of 130 adequate and 24 inadequate samples were collected from patients participating in drug withdrawal therapy. To obtain saliva from the oral cavity, a newly developed collection device (Clin Rep), consisting of a treated cotton roll and a centrifugation vial with a filter inset, was tested. For the preparation of a purified solution of urine, liquid extraction was used. Solid phase extraction was utilized to prepare the saliva samples. For the detection by gas chromatography-mass spectrometry, an appropriate derivatization was necessary using N-methyl-N-(trimethylsilyl)trifluoroacetamide. The retention times were compared with defined standard solutions. The obtained mass spectra showed a characteristic fragmenting pattern and offered a reliable identification. RESULTS: The concordance of the analytic results of the saliva samples with urine was 93% for a decision limit of 100 ng/mL and 98% for a decision limit of 300 ng/mL (DHHS opiate cutoff) in urine. CONCLUSIONS: Saliva, of adequate amount in 85% of samples, may be appropriate for analysis of drugs of abuse.

Determination of lamotrigine, carbamazepine and carbamazepine epoxide in human serum by gas chromatography mass spectrometry.

A method for the identification and quantification of the antiepileptics lamotrigine, carbamazepine and carbamazepine epoxide (active metabolite) in human serum is described. In refractory epilepsy the combination of carbamazepine and lamotrigine was recently developed to a modern therapeutic concept. The goal of this paper is therapeutic drug monitoring (TDM) of these substances. Serum was extracted with a quick precipitation method using a modified commercial extraction-kit and analysed by gas chromatography mass spectrometry (GC-MS). A gas-chromatographic temperature-pressure programme was developed that allowed the determination of lamotrigine by gas chromatography mass spectrometry. A reference spectrum of pure lamotrigine is herewith published for the first time. A new mass spectra library was created to support the identification of the antiepileptics in human serum. In the Specified Ions Monitoring mode (SIM) a detection limit below the therapeutic range and recoveries above 90% were obtained. Comparison of results obtained by GC-MS or a commercially available high performance liquid chromatographic (HPLC) method (for lamotrigine) and a fluorescence polarisation immunoassay (FPIA) (for carbamazepine) from spiked serum samples showed disagreement of no more than 10% between the methods and demonstrated the accuracy of the new method. In addition, quantitative determinations of these antiepileptics in samples from patients under anticonvulsive therapy showed a strong linear correlation with r2 = 0.961 for carbamazepine and r2 = 0.964 for lamotrigine. Only two from a total of 46 results differed by more than 10%. Our method for quantifying lamotrigine in serum seems to be highly specific and capable of measuring lamotrigine in patients on single therapy, as well as on add-on therapy with carbamazepine or carbamazepine epoxide. No interference from other coadministered drugs was detected.

Purification of human and rat kidney aldose reductase.

In the present study we report on a rapid two-step affinity chromatographic procedure to purify aldose reductase from human and rat kidney papilla and inner medulla. This enzyme, which is responsible for sorbitol formation in the kidney, was purified 145-fold from rat and 76-fold from human kidneys by consecutive Blue Sepharose and Matrex Orange chromatography. SDS-PAGE showed a single band of 38 kD for the human enzyme and a doublet of similar molecular weight for the rat kidney aldose reductase. The enzyme was characterized by substrate specificity and kinetic constants found identical to that of other organs purified previously.

Mechanized toxicological serum tests in screening hospitalized patients.

A spectrum of quantitative and qualitative methods was adapted to the RA-1000/RA-XT selective analyser for the purpose of excluding or detecting common types of intoxication in the emergency laboratory of our primary care community hospital. Ethanol and salicylates (measured photometrically) and acetaminophen (measured immunologically by EMIT tox) were quantitatively analysed in serum. immunological group tests (EMIT tox) for barbiturates, benzodiazepines, tricyclic antidepressants and related compounds were used for qualitative analysis. Well established clinical chemical methods (aspartarte aminotransferase, alanine aminotransferase, creatine kinase, pseudocholinesterase, glucose and lactate) were applied to the serum samples using the same selective analyser. Within and between run precision, accuracy, recovery and detection ranges (linearity) fulfilled the recommendations of forefield toxicological analysis for all methods. Ethanol (g/l), measured photometrically with the RA-1000 analyser, agreed with the reference method (headspace gas-chromatography) with a correlation coefficient greater than 0.99 (y = 0.06 + 0.98x). Acetaminophen and salicylates showed correlation coefficients greater than 0.94 and greater than 0.99, when compared with manual colorimetric procedures (acetaminophen (mg/l): y = -3.22 + 0.896x; salicylates (mg/l): y = -2.1 + 1x). Qualitative group tests for barbiturates, benzodiazepines and tricyclic antidepressants measured with the RA-1000 analyser were in good agreement with the EMIT single test procedure. The ranges of the quantitative methods allowed quantification of analytes from therapeutic (non-toxic) to very high levels in undiluted samples (ethanol 0.05 up to 4 g/l; salicylates 32 up to 1200 mg/l and acetaminophen 1.9 up to 200 mg/l). The low detection limits of the qualitative tests allowed the recognition of compounds in plasma that were present in low concentrations and/or displayed only minor reactivity with the antibodies provided by the EMIT tox test kits. As a consequence, decision limits for all three group tests in serum were lowered to near the detection limit: (table: see text) For quantitative tests the lower limits of quantification were: (table: see text) The working reagents were stable for at least 14 days at 4-8 degrees C. Calibration curves were stable over the expiration period of reconstituted original reagents (6-12 weeks), also when working reagents were prepared in aliquots from stored reconstituted reagents. Application of the newly adapted programme to serum samples of nearly two hundred patients showed it to be suitable for screening patients in which intoxication is suspected or needs to be excluded.

Overestimation of albumin in heparinized plasma.

Monochromatic measurement of albumin by the bromcresol green method leads to overestimation of albumin in the presence of heparin. The interference seems to be caused by fibrinogen, which, in the presence of heparin, produces an almost constant increase of the measuring signal over the whole range of suitable wavelengths (550 to 700 nm). The albumin overestimation can be eliminated by using a bichromatic modification. An application of this procedure for the automated analyzer RAXT, RA-1000 (Bayer Diagnostik, Technicon) is presented. We recommend using only bichromatic methods for albumin determination with bromcresol green to avoid unexpected analytical artifacts.

Localization and regulation of the renal kallikrein kinin system: possible relations to renal transport functions.

The complete renal kallikrein kinin system has recently been localized in defined nephron segments. Kallikrein was found to be formed and secreted by connecting tubule cells in the late distal convoluted tubule, whereas kininogen and a novel kininase were located in collecting tubules. Kinins were shown to act on collecting tubule as well as medullary interstitial cells and the renal vasculature. The literature on interactions of this system with renal sodium transport is conflicting. Renal and urinary kallikrein was found to be increased under sodium restricted conditions, whereas kinin has a diuretic and natriuretic effect in the collecting tubule, when added from the basolateral surface. On the other hand renal kallikrein activity and connecting tubule cell morphology change in parallel with dietary potassium load indicating a coupling to potassium secretion. The possible role of the renal kallikrein kinin system in regulating collecting tubule function by tubular and vascular effects is outlined in spite of many open questions which remain to be answered.

Quantification of kininogen in human renal medulla.

Renal kininogen was detected in human medullary tissue as well as human medullary tubule suspensions. After treatment with pig pancreatic kallikrein or human renal cortical homogenate liberated kinin was measured by bradykinin radioimmunoassay. In the absence of inhibitors kinins were degraded by kininases located in the same part of the kidney. Several known inhibitors of kininase I and II did not inhibit this activity. Endogenous medullary kininase was inhibited by preincubation of homogenates at 56 degrees C for one hour or by addition of 0.25 mmol/l HgCl2. Under these conditions endogenous medullary kinin release amounted to 9-26 nmol/g protein. The action of renal cortical kininogenase on kinin formation from papillary kininogen was completely inhibited by addition of 1 mumol/l aprotinin. Kininogen examined in renal tubule suspensions revealed an increase in amount per g protein compared to homogenates, confirming the tubular localization of renal kininogen.

Kallikrein (kininogenase) in the mouse nephron: effect of dietary potassium.

Kininogenase activity of kallikrein was measured in microdissected mouse nephron segments using kininogen from dog plasma and a radioimmunoassay for bradykinin. When single nephron segments were examined, results showed a large scatter. This was found to be due to heterogeneity of distal convoluted tubules (DCT) from different nephrons, since replicate measurements in pools of DCT structures did not show this degree of variation. Nearly 20% of activity was accessible to extracellular substrate when freshly dissected segments were incubated in isoosmotic media. Freezing and thawing which markedly releases activity of intracellular enzymes, did not significantly elevate kininogenase activity. On the other hand deoxycholate and trypsin treatment increased tubular kininogenase activity in an additive fashion. A detailed analysis of microdissected tubule fragments revealed that kallikrein is concentrated in late distal convoluted tubule before entering a branching point (connecting tubule). In contrast initial portions of distal convoluted tubules and cortical collecting tubules contained only little kallikrein activity. Potassium rich diet increased basal and total activity 5-fold, when compared to a potassium poor diet.

Quantitative assessment of the binding of acetaminophen metabolites to mouse liver microsomal phospholipid.

Phospholipids were quantitatively extracted from microsomes and separated by an h.p.l.c. gradient system with a solvent mixture of n-hexane/n-propanol/water/acetic acid. In a model reaction using horseradish peroxidase/H2O2 in order to activate acetaminophen and inactivated microsomes as target, a covalent binding of 10 nmol drug metabolite per mg microsomal lipid was found. In isolated intact microsomes from methylcholanthrene-pretreated male albino mice, a binding of 0.1 nmol acetaminophen metabolite per mg phospholipid was determined while the binding of metabolites to protein amounted to 3 nmol/mg. The results demonstrate that in mouse liver microsomes metabolizing acetaminophen, about one out of 10(4) phospholipid molecules is modified.