Drug screening of hair by liquid chromatography-tandem mass spectrometry.

Hair has become an important matrix for drug analysis, complementary to blood and urine as a matrix. A prolonged detection window makes hair analysis suitable for the detection of exposure to illegal and medicinal drugs for periods up to 12 months. In the present study, a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for drug screening in hair was developed and validated. To 20 mg of hair, 0.45 mL of acetonitrile/25 mM formic acid (5:95 v/v) and 50 microL of deuterated internal standards were added, and the sample was incubated in a water bath at 37 degrees C for 18 h. LC separation was achieved with a Zorbax SB-Phenyl column (2.1 x 100 mm, 3.5-microm particle). Mass detection was performed by positive ion mode electrospray LC-MS-MS and included the following drugs/metabolites: nicotine, cotinine, morphine, 6-monoacetylmorphine, codeine, amphetamine, methamphetamine, 3,4-methylenedioxymeth-amphetamine, cocaine, benzoylecgonine, 7-aminonitrazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, oxazepam, diazepam, alprazolam, zopiclone, zolpidem, carisoprodol, meprobamate, buprenorphine, and methadone. Within- and between-assay relative standard deviations varied from 2.0% to 12% and 2.7% to 15%, respectively. The accuracies were in the range of -24% to 16%, and recoveries ranged from 25% to 100%. The LC-MS-MS method proved to be simple and robust for the determination of drugs in hair. It has been used for authentic samples in our laboratory in the past year.

Rapid quantification of buprenorphine-glucuronide and norbuprenorphine-glucuronide in human urine by LC-MS-MS.

A liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed and validated for the determination of buprenorphine-glucuronide (BUP-G) and norbuprenorphine-glucuronide (NBUP-G) in human urine. The method included a dilution step followed by filtration through a Mini-Uniprep Filter and direct injection onto the LC column. The analytes were quantified in multiple reactions monitoring mode using one transition ion. Norbuprenorpine-d(3) (NBUP-d(3)) was used as the internal standard. The concentration ranges were 6-161 ng/mL for BUP-G and 12-295 ng/mL for NBUP-G. Recoveries determined after filtration for the analytes were 75%. The between-day precision of the method was in the range of 4.8-11%. The limits of quantification were found to be 4.6 ng/mL for BUP-G and 11.8 ng/mL for NBUP-G. Approximately 1000 samples from law enforcement, prison inmates, probation services, and hospitals were analyzed by the presented method. The ratios of drug glucuronides versus creatinine were calculated for a selection of samples (n = 151), where there was information on treatment with buprenorphine between 16 and 20 mg/day. The majority (86%) of the samples had a ratio of BUP-G/creatinine below 570 microg/g, and 76% of the samples had NBUP-G/creatinine lower than 1060 microg/g. The LC-MS-MS method proved to be robust and specific for the determination of BUP-G and NBUP-G in urine.

Alterations in capillary permeability in the lower limb of patients with chronic critical limb ischaemia and oedema.

BACKGROUND: Oedema formation in lower limbs of patients with chronic critical limb ischaemia (CLI) is a common clinical feature. The rate of fluid filtration through the capillary wall depends on the capillary permeability, i.e. capillary filtration coefficient (CFC). In order to elucidate the pathogenesis of this ischaemic oedema, CFC was measured in the limbs with CLI and oedema and was compared with CFC measurements both in the contralateral sides and in the lower limbs of a control group. PATIENTS AND METHODS: Eleven women and 4 men, with a mean age of 75 +/- 8.8 years, with unilateral CLI and leg and foot oedema were included. Leg and foot volume was measured with water displacement volumetry (WDV). CFC was measured in both limbs by strain-gauge plethysmography using a double-stranded mercury in silicone strain gauge around the middle portion of the foot. As a control group, 8 patients, 5 women and 3 men, with a mean age of 77 +/- 7.6 years with a proximal femur fracture were included and the CFC in this group was measured in the foot of the non-fractured limb. RESULTS: Mean CFC in the limbs with CLI and oedema was 0.0036 +/- 0.001 ml/min.100 ml.mmHg, significantly greater than both the contralateral limbs (0.0019 +/- 0.0003 ml/min.100 ml.mmHg, p < 0.01) and mean CFC in the control limbs (0.0017 +/- 0.0002 ml/min.100 ml.mmHg, p < 0.003). There was a mean volume difference of 13 +/- 9% between limbs with CLI and contralateral sides measured by WDV. There was no significant correlation between total leg-foot volume and CFC (p > 0.05). CONCLUSION: CFC in the ischaemic limb was twice as great as both the contralateral side and the limbs of the control group. It is therefore concluded that an increased CFC is probably one of the important factors in the development of this ischaemic oedema.

The concentration of protein-compounds in interstitial tissue of patients with chronic critical limb ischaemia and oedema.

BACKGROUND: Many of chronic critical limb ischaemia (CLI) patients have distal leg and foot oedema. Previous electronmicroscopic studies have shown that chronic severe ischaemia may cause hypoxic damage of the capillary endothelium, including morphological changes i.e. multiplicated/thickened basal lamina, and formation of interendothelial gaps. To assess the functional consequences of these morphologic derangements, where proteins can leak through, we investigated the composition of the interstitial fluid in oedematous ischaemic limbs. PATIENTS AND METHODS: Nine female and 3 male patients with a mean age of 79 +/- 7.9 years were included. All had unilateral CLI and peripheral pitting oedema. Leg and foot volume was measured with water displacement volumetry. Blister suction technique was used to collect subcutaneous interstitial fluid. The concentration of albumin, transferrin, immunoglobulin G and alpha 2-macroglobulin in plasma and blister fluid was measured by immunoturbidimetry. Nine patients, 8 women and 1 man with a mean age of 83 +/- 5.5 years with a proximal femur fracture served as an age-matched control group. RESULTS: The mean concentration of albumin in blister fluid was significantly lower in the patients, whereas the mean concentration of alpha 2-macroglobulin in blister fluid did not differ between patients and controls. Mean ratio between concentrations in blister and serum of albumin, transferrin and immunoglobulin G in the limbs with CLI and oedema were significantly lower than respective values in the control group. However, there was no significant difference in the ratio of alpha 2-macroglobulin between these groups. CONCLUSION: A higher transcapillary concentration gradient for proteins in CLI limbs signifies an increase in the net osmotic pressure gradient across the capillary wall, which may be a potential oedema limiting factor.

The distribution of oedema in the lower limb of patients with chronic critical limb ischaemia: a study with computed tomography.

BACKGROUND: A substantial number of patients with chronic critical limb ischaemia (CLI) have considerable oedema at the distal leg and foot of non deep venous thrombosis origin. The primary aim of the present study was to quantify the distribution of oedema in the different tissues of the leg and foot by applying computed tomography and planimetry. The interstitial fluid hydrostatic pressure (Pif) in the subcutaneous tissue was measured to evaluate the effect of oedema on local tissue pressure. PATIENTS AND METHODS: Six men and 12 women with unilateral CLI and peripheral pitting oedema were included. Cross sectional areas (CSA) of subcutaneous tissue, muscle and bone were measured by computer tomography combined with planimetry to assess the distribution of oedema within the soft tissues. Pif was measured by "wick-in-needle" technique. RESULTS: The median total CSA of soft tissue, subcutaneous and muscle tissues at the foot level were respectively 17%, 34% and 9% greater in the limbs with CLI compared to the contralateral limb (p < 0.001). At ankle level these differences were 13%, 30% and 4%, respectively (p < 0.001). At the level of the calf these differences were not significant. Mean Pif in the limbs with CLI was 0.3 mmHg, significantly higher than in limbs without CLI (-1.8 mmHg), (p < 0.003). CONCLUSION: The study verified oedema of considerable magnitude at the ankle and foot. The great part of the oedema was located within the subcutaneous tissue, which was associated with a relatively moderate, but significant increase in Pif confirming the high compliance of the subcutaneous tissue. The combination of the excessive fluid and increased Pif in the interstitial tissue might aggravate the microcirculation. The aetiology of oedema formation is probably multifactorial.

No case of COX-1-related aspirin resistance found in 289 patients with symptoms of stable CHD remitted for coronary angiography.

OBJECTIVE: To assess the prevalence of a lacking aspirin effect on cyclooxygenase-1 (COX-1) ("aspirin resistance") in patients with symptomatic, stable coronary heart disease (CHD) using test methods directly reflecting inhibition of COX-1. MATERIAL AND METHODS: Arachidonic acid (AA)-induced platelet aggregation and plasma thromboxane B2 (TXB2) were determined twice 3 weeks apart - prior to elective coronary angiography - in 289 patients on 75 or 160 mg aspirin daily, all prompted to take aspirin before testing. Subjects who demonstrated lacking any effect of aspirin (>/=20 % AA-induced aggregation) on one or both occasions were later given a third test. Forty-two patients not taking aspirin were used as TXB2 controls. RESULTS: Eleven (3.8 %) had aggregation > or = 20 % in at least one of the two initial tests, but only two on both occasions. During the third test, all 11 patients had aggregation <20 %. The TXB2 distributions in controls and study patients differed markedly (mean 173 versus 19 pg/mL). Taking 45 pg/mL as the TXB2 cut-off level, sensitivity and specificity for detecting subjects taking aspirin were 90 % and 89 %, respectively. The area under the ROC curve was 0.96. CONCLUSION: Repeated AA-induced platelet aggregometry showed that COX-1 could be blocked by low-dose aspirin in all 289 tested patients, suggesting that aspirin resistance is rare in patients with stable CHD.

Patterns of skin flowmotion in the lower limbs of patients with chronic critical limb ischaemia (CLI) and oedema.

OBJECTIVE: To study flowmotion (FM) in lower limbs with critical limb ischaemia (CLI) and oedema and to elucidate FM patterns when skin viability is threatened. MATERIAL AND METHODS: Fourteen patients with unilateral CLI and oedema and two control groups were included - one consisting of 10 healthy participants and the other nine patients with unilateral CLI without oedema. Laser Doppler was used to evaluate the foot skin microcirculation simultaneously at four different areas, with the limbs in supine and dependent position. FM was expressed using fast Fourier transformation (FFT) as low frequency (LF) and high frequency (HF) waves and their respective FFT-powers. RESULTS: All patients with CLI, both with and without oedema, showed HF waves in both diseased and contralateral limbs. These were absent in healthy controls. There were no regional differences in frequency in the critically ischaemic feet (with and without oedema) and between ischaemic and their contralateral feet. Changing the position of ischaemic limbs from supine to dependency had no significant effect on the frequency, while a significant increase of the median FFT-powers of LF and HF waves at the pulp of the first toe was observed. This manoeuvre resulted in decrease of the median FFT-powers of LF in healthy controls. CONCLUSIONS: HF waves are associated with CLI. Ischaemia also appears to influence the FFT-power of each frequency domain. Ischaemic oedema does not seem to affect the pattern of FM in the foot of patients with CLI.

The role of the veno-arteriolar reflex (VAR) in the pathogenesis of peripheral oedema in patients with chronic critical limb ischaemia (CLI).

BACKGROUND AND AIMS: Relatively many patients with chronic critical limb ischaemia (CLI) have oedema in the afflicted limb. Previous studies in these patients indicate derangement in the Starling forces governing transcapillary fluid balance. An impaired veno-arteriolar reflex (VAR) may cause an increase in capillary pressure and hence increased filtration pressure resulting in oedema. The aim of the present study was to investigate VAR in patients with CLI to come to a better understanding of the pathogenesis of ischaemic oedema. MATERIAL AND METHODS: Sixteen patients (mean age of 78+/-9.4 years) with unilateral CLI and oedema were included. There were two control groups of similar age, one consisted of 8 patients with unilateral CLI but without oedema and the other of 9 healthy subjects. Laser Doppler fluxmeters were used to evaluate the foot skin microcirculation, with the limbs in supine as well as in the dependent position, simultaneously in 4 different areas: the pulp of the first toe (Sitel), at the level of the second metatarsal body (Site 2), at the anterolateral part of ankle (Site 3) and the pulp of the first toe of the contralateral limb (Site 4) as reference. Laser Doppler flux (LDF) values (expressed in perfusion unit, PU) were recorded with the foot dependent (PUd), in the horizontal position (PUh), and the orthostatic response (OR) was calculated at all measuring sites as PUd/PUh. RESULTS: In none of the measured sites there was a significant difference in OR between the limbs with CLI and oedema and the limbs with CLI but without oedema. The median OR of CLI limbs at site 1 [2.5 (0.61-8.96)] was greater than at sites 2 [0.99 (0.46-2.38), p < 0.01] and 3 [0.95 (0.68-10.31), p < 0.04] respectively, while the differences in OR between site 2 and 3 were not significant. The median OR in the limbs of healthy controls at sites 1, 2 and 3 were 0.58 (0.37-1.43), 0.54 (0.28-1.33) and 0.51 (0.34-0.91), respectively. There were no significant differences in OR between sites 1, 2 and 3 of the control group. The OR of CLI at site 1, 2 and 3 were significantly greater than the corresponding sites in the healthy subjects (p < 0.001, p < 0.008, p < 0.001). CONCLUSIONS: The VAR is disturbed in limbs with CLI, both in the ones with and without oedema. There were regional differences in the OR in these ischaemic feet but there were no differences in OR between those with and without oedema. Thus, disturbances in VAR may play a role in the development of ischaemic oedema, but is probably not the only causative factor.

Vascular and stromal features in the skin of the lower limb in patients with chronic critical limb ischaemia (CLI) and oedema.

OBJECTIVE: peripheral oedema is often observed in limbs affected by chronic critical limb ischaemia (CLI) and is mainly subcutaneous in distribution. Previous work has shown that capillary filtration coefficient (CFC) in limbs with CLI and oedema was twice as great as that in the contralateral limb. These changes might be due to morphological changes. Transmission electron microscopy (TEM) was used to examine the morphological features of the capillary walls and surrounding stromal tissues in the skin of these limbs. MATERIAL AND METHODS: eight patients with unilateral CLI and peripheral pitting oedema (four men, four women, a mean age of 81+/-6.9 years) was studied. Skin biopsies were taken from the pulp of the first toe, interdigital space between the first and second digits and dorsal part of forefoot just prior to amputation. RESULTS: stromal oedema and dilated capillaries were most prominent in the distal part of the foot. Some of the capillaries were filled with blood cells and some were empty. The endothelium of the dilated vessels was elongated and distended. In some patients a number of capillaries were collapsed with degenerate endothelial cells. <<<>>>, i.e. large openings, were found between the elongated oedematous endothelial cells. The basal lamina was thickened in all patients. Stromal haemorrhage and degeneration were seen in approximately 50% of patients. CONCLUSION: CLI causes ultrastructural changes in the capillary endothelium and surrounding stroma. The presence of large gaps between endothelial cells as well as an increased capillary pressure may enhance transcapillary transudation, and are most likely the causative factors in the formation of the ischaemic oedema. The stromal haemorrhage as well as degeneration probably signifies a terminal stage of CLI.

Transcapillary forces and the development of oedema in the lower limb of patients with chronic critical limb ischaemia (CLI).

OBJECTIVE: factors regulating transcapillary fluid transport were investigated to elucidate the causes of oedema in CLI. MATERIAL: sixteen patients, 6 men and 10 women (mean age of 79+/-10.3 years) with unilateral CLI and peripheral pitting oedema. METHODS: measurements were performed in both limbs. Interstitial fluid was collected by applying blister suction cups on the dorsolateral part of the foot and colloid osmotic pressure of this fluid (COP if) was measured in a colloid oncometer. Plasma colloid osmotic pressure (COP pl) was obtained from venous blood. Interstitial fluid pressure (P if) was measured by wick-in-needle technique. RESULTS: mean COP if in the limbs with CLI was 2.3 S.D. 0.5 mmHg, significantly lower than in the limbs without CLI (3.1 S.D. 0.7 mmHg, p<0.0001). Mean COP pl was 21.1 S.D. 1.8 mmHg, which was lower than in healthy controls. Mean plasma albumin concentration was 30 S.D. 6 g/l which was lower than the reference values. Mean P if in the limbs with CLI was 0.7 S.D. 1.6 mmHg, significantly higher than in the limbs without CLI (-1.4 S.D. 1.4 mmHg, p<0.0001). The calculated mean reabsorption pressure (P r) in the limbs with CLI was 19.6 S.D. 1.7 mmHg, significantly higher than in the contralateral limbs (16.7 S.D. 2.1 mmHg, p<0.001). CONCLUSION: a low plasma albumin concentration in patients with CLI agrees with the reduction in COP pl but cannot explain the oedema formation, since it is unilateral. The high P r may cause a high transcapillary filtration pressure, resulting in a relatively great net filtration and subsequent oedema formation.

Oedema in the lower limb of patients with chronic critical limb ischaemia (CLI).

OBJECTIVE: approximately 70% of patients with chronic critical limb ischaemia (CLI) show clinical signs of oedema in the distal leg and foot. The primary aim of the present investigation was to quantify this oedema. In addition we investigated whether oedema formation could be due to deep venous thrombosis (DVT). METHODS: fifteen patients with unilateral CLI and oedema were studied, four males and 11 females, with a mean age of 77+/-10.3 years. Water displacement volumetry (WDV) was used to measure limb volume. Colour duplex ultrasound (CDU) and venous occlusion plethysmography (VOP) were applied to exclude functionally significant DVT. Blood chemistry was analysed to screen for some causative factors of generalised oedema formation. RESULTS: the mean volume of the limbs with CLI was 9% greater than the contralateral limbs (1279+/-325 ml vs. 1179+/-298 ml). None of the patients had functionally significant DVT. The mean plasma albumin concentration was reduced at 28.5+/-6.6 g/l. CONCLUSION: a significantly reduced plasma albumin concentration cannot be regarded as a causative factor, since the oedema is unilateral. The aetiology of oedema formation is probably multifactorial, and further investigations are under progress to elucidate relevant pathogenetic factors.