Resonance Rayleigh scattering technique as a detection method for the RP-HPLC determination of local anaesthetics in human urine.

A highly selective and sensitive method of reversed phase high-performance liquid chromatography (RP-HPLC) coupled with resonance Rayleigh scattering (RRS) was developed for the determination of procaine, bupivacaine and tetracaine. Separation of three local anaesthetics was achieved at 35 °C on a C18 column. The mobile phase was 30: 70 (v/v) acetonitrile/triethylamine-phosphoric acid buffer (pH 2.9) at flow rate of 0.3 mL/min. The RRS detection was conducted by taking advantage of the strong RRS enhancement of the local anaesthetics with erythrosine reaction in an acidic medium. Under optimum conditions, the limit of detection (S/N = 3) values were in the range of 2.4-11.2 ng/mL. Recoveries from spiked human urine samples were 95.8%-104.5%. The proposed method applied to the determination of local anaesthetics in human urine achieved satisfactory results. In addition, the mechanism of the reaction is fully discussed. Copyright © 2016 John Wiley & Sons, Ltd.

On-line preconcentration strategy for the simultaneous quantification of three local anesthetics in human urine using CZE.

The simultaneous determination of usually employed anesthetics (procaine, lidocaine, and bupivacaine) has been developed and validated using CE with ultraviolet detection at 212 nm. The separation of these three drugs has been achieved in less than 7 min, using a temperature of 25ºC and 25 kV, with a 150 mM citrate buffer (pH 2.5) as BGE. Field-amplified sample injection (FASI) has been used for on-line sample preconcentration. Ultrapure water and ACN 50/50 (v/v) mixture gave the greatest enhancement factor when it was employed as an injection solvent. Injection voltage and time were optimized, being 13 kV and 13 s, the optimum values, respectively. To avoid the possible irreproducibility associated with the electrokinetic injection, an internal standard such as tetracaine, was employed. The instrumental detection limits (LOD S/N = 3) for the compounds ranged between 2.6 and 7.0 µg L(-1) and the quantitation limits (LOQ S/N = 10) between 37.8 and 55.9 µg L(-1) . The detection limits obtained in real human urine samples ranged between 55.2 and 83.6 µg L(-1) and the quantitation limits between 196.0 and 276.0 µg L(-1) . The proposed method has demonstrated its applicability to the analysis of these local anesthetics in urine samples without any pretreatment, allowing the rapid determination of these target analytes.

Molecularly imprinted polymer in microextraction by packed sorbent for the simultaneous determination of local anesthetics: lidocaine, ropivacaine, mepivacaine and bupivacaine in plasma and urine samples.

This study presents the use of molecularly imprinted polymer (MIP) as packing material for microextraction by packed syringe (MEPS) to achieve higher extraction selectivity. Pentycaine was used as template for MIP. Development and validation of the determination of lidocaine, ropivacaine, mepivacaine and bupivacaine in human plasma and urine samples utilizing MIP-MEPS and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were carried out. The MEPS MIP-cartridge could be used for 100 extractions before it was discarded. The extraction recovery ranged from 60 to 80%. The correlation coefficients values were >0.999 for all assays using lidocaine, ropivacaine, mepivacaine and bupivacaine in the calibration range 5-2000 nmol/L. The accuracy of the studied compounds, given as a percentage variation from the nominal concentration values, ranged from -4.9 to 8.4% using plasma and urine samples. The between-batch precision, given as the relative standard deviation, at three different concentrations (quality control samples) was ranged from -4.7 to 14.0% and from 1.8 to 12.7% in plasma and urine, respectively. The lower limit of quantification and limit of detection of the studied substances were 5.0 and 1.0 nm, respectively.

Structural elucidation of phase I and II metabolites of bupivacaine in horse urine and fungi of the Cunninghamella species using liquid chromatography/multi-stage mass spectrometry.

RATIONALE: Bupivacaine is a local anaesthetic prohibited in equine sports. It is highly metabolized in the horse but a thorough description of its metabolite profile is lacking. An administration study should find appropriate analytical targets for doping control. Furthermore, knowledge of an in vitro system for production of metabolites would be beneficial. METHODS: Marcain® (bupivacaine hydrochloride) was administered subcutaneously to a horse and urine samples were collected. In vitro metabolic systems consisting of the fungi Cunninghamella elegans and Cunninghamella blakesleeana were incubated with bupivacaine and bupivacaine-d(9). Samples were analyzed directly after dilution or cleaned up using liquid-liquid extraction. Separation was achieved with liquid chromatography. Mass spectrometric analysis was performed using positive electrospray ionization with both a tandem quadrupole and an ion trap instrument using MS(n) and hydrogen/deuterium exchange. RESULTS: In horse urine, seven phase I metabolites were found: 3'- and 4'-hydroxybupivacaine, N-desbutylbupivacaine, two aliphatically hydroxylated metabolites, one N-oxide, and dihydroxybupivacaine. Sulfated hydroxybupivacaine and glucuronides of 3'- and 4'-hydroxybupivacaine and of dihydroxybupivacaine were also detected. All these metabolites were previously undescribed in the horse, except for 3'-hydroxybupivacaine. 3'- and 4'-Hydroxybupivacaine were designated as appropriate targets for doping control. Interestingly, all the equine phase I metabolites were also detected in the samples from C. elegans and C. blakesleeana. CONCLUSIONS: The qualitative aspects of the metabolism of bupivacaine in the horse have been investigated with many novel metabolites described. The fungi C. elegans and C. blakesleeana have proven to be relevant models for mammalian metabolism of bupivacaine and they may in the future be used to produce analytical reference materials.

[Decomposition kinetics of bupivacaine in biological specimens].

OBJECTIVE: To study on the decomposition kinetics of bupivacaine in brain, blood and urine, which were collected from dogs executed by bupivacaine and stored in different conditions. METHODS: Dogs were given arachnoid cavity anesthesia with bupivacaine. Then the brain, blood and urine were collected and divided equally to three groups stored in 20, 4 and -20 degrees C respectively. The concentrations of bupivacaine at different days were determined by the GC. The equation and half-time period of decomposition kinetics were imitated and calculated with WinNolin program. RESULTS: The decomposition kinetics of bupivacaine in the dogs' brain, blood and urine were fit to the first order kinetics. The common equation was lgC = lgCo-kt/2.303 and k was the decomposition constant of first order reaction. CONCLUSION: Bupivacaine in the brain, blood and urine specimens were found to be decomposed at various environments for storage. The higher temperature for storage, the faster of decomposition reaction.

Liquid-phase microextraction combined with high-performance liquid chromatography for the determination of local anaesthetics in human urine.

A simple liquid-phase microextraction (LPME) device combined with high-performance liquid chromatography (HPLC) is presented for the simultaneous analysis of local anaesthetics, lidocaine, bupivacaine, and tetracaine, from human urine sample. An organic solvent showed good compatibility with the mobile phase of the HPLC, o-dibutyl phthalate, was selected. Local anaesthetics are extracted from 6 ml of the feed aqueous solution and human urine sample into a water-immiscible organic solvent suspended at the needle tip of the microsyringe, then the organic solvent was directly introduced to a reversed-phase HPLC system. The kind of the organic extraction solvent, the stirring rate, the pH value of the aqueous feed solution, and the extraction time have been discussed. Under the optimized extraction conditions, high enrichment factors (more than 86.0-fold) and significant sample clean-up for all of studied local anaesthetics were achieved within 30 min. The detection limits (lower than 0.05 microg/ml) were comparable with previously reported gas chromatography methods. This method was applied to specimen of patient who was treated with extradural anaesthesia of lidocaine, bupivacaine, and tetracaine, and revealed that simultaneous determination of above three local anaesthetics in human urine was possible.

Spinal anesthesia for arthroscopic knee surgery.

BACKGROUND AND OBJECTIVE: The purpose of the study was to compare the effects of adding 50 microg of morphine, 25 microg of fentanyl or saline to 6 mg of hyperbaric bupivacaine on postoperative analgesia and time to urination in patients undergoing arthroscopic knee surgery under spinal anesthesia. METHODS: The study was designed in a prospective, randomized, double-blinded and placebo-controlled manner. Sixty ASA I-II patients were randomized into the following three groups: Group BM: 6 mg of bupivacaine and 50 microg of morphine, Group BF: 6 mg of bupivacaine and 25 microg of fentanyl, and Group BS: 6 mg of bupivacaine and saline. Selective spinal anesthesia was performed in a lateral decubitus position, with the operative knee dependent for 10 min. RESULTS: In all groups satisfactory anesthesia was provided during the operation. There was a statistically significant difference between all the groups in times to voiding [Group BM 422 +/- 161 min; Group BF 244 +/- 163 min; Group BS 183 +/- 54 min (mean +/- SD)]. The incidence of pruritus was significantly greater in Group BM (80%) and BF (65%) in comparison with Group BS (no pruritus) (P < 0.05). The incidence of nausea was significantly increased in Group BM (35%) in comparison with Group BF (10%) and Group BS (P < 0.05). Analgesic consumption was significantly greater in Group BS in comparison with Groups BM and BF (P < 0.01). CONCLUSIONS: We conclude that during spinal anesthesia even mini-dose intrathecal morphine is not acceptable for outpatient surgery due to side-effects, especially severely prolonged time to urination.

Nonaromatic hydroxylation of bupivacaine during continuous epidural infusion in man.

Less than 11% of the dose of bupivacaine could be accounted for in urine from 10 patients receiving continuous epidural infusions. HPLC analysis of metabolites confirmed (S)-bupivacaine was more extensively metabolised than (R)-bupivacaine, and dealkylation was the predominant metabolic pathway although co-elution of metabolites made quantitation difficult. The percentage of (S)-2',6'-pipecoloxylidide and co-eluting metabolites excreted relative to (R)-2',6'-pipecoloxylidide from three patients was 0.32+/-0.05, while for seven patients it was 1.28+/-0.09. Conversely, the percentage of (S)-3'-hydroxy bupivacaine and co-eluants excreted relative to (R)-2',6'-pipecoloxylidide from the three patients (1.76+/-0.48) was greater than the seven patients (0.19+/-0.09). Urinary metabolites were analysed for evidence of aliphatic hydroxylation of bupivacaine. Chiral liquid chromatography-mass spectrometry (LC-MS) on an alpha(1)-glycoprotein column at pH 7 used hydroxylamine acetate as the volatile mobile phase. Compounds tentatively identified as hydroxybupivacaines by MRM were verified by their product ion spectra in a subsequent MS-MS run. Eighteen oxygenated metabolites of bupivacaine were detected, half of which were hydroxylated on nonaromatic groups. Equal numbers of mono- and dihydroxybupivacaines were excreted. There was no evidence to suggest the presence of (S)-4'-hydroxybupivacaine, 2'-hydroxymethylbupivacaine, 3'-hydroxy-2',6'-pipecoloxylidide or a piperidone. The metabolite previously identified as (S)-4'-hydroxybupivacaine was not hydroxylated on the xylyl group.

Determination of bupivacaine and metabolites in rat urine using capillary electrophoresis with mass spectrometric detection.

A method using capillary electrophoresis-mass spectrometry (CE-MS) was developed for the structural elucidation of bupivacaine and metabolites in rat urine. Prior to CE-MS analysis, solid-phase extraction (SPE) was used for sample cleanup and preconcentration purposes. Exact mass and tandem mass spectrometric (MS/MS) experiments were performed to obtain structural information about the unknown metabolites. Two instruments with different mass analyzers were used for mass spectrometric detection. A quadrupole time-of-flight (Q-TOF) and a magnetic sector hybrid instrument were coupled to CE and used for the analysis of urine extracts. Hydroxybupivacaine as well as five other isomerically different metabolites were detected including methoxylated bupivacaine.

Effect of ciprofloxacin on the pharmacokinetics of ropivacaine.

OBJECTIVE: To assess the effect of ciprofloxacin on the pharmacokinetics of ropivacaine. METHODS. In a double-blind, randomised, cross-over study, nine healthy volunteers were treated for 2.5 days with 500 mg oral ciprofloxacin or placebo twice daily. On day 3, they received a single dose of 0.6 mg/kg ropivacaine intravenously over 30 min. Ropivacaine, 3-hydroxyropivacaine (3-OH-ropivacaine), and (S)-2',6'-pipecoloxylidide (PPX) in venous plasma and urine were measured for up to 12 h and 24 h, respectively. RESULTS: Ciprofloxacin decreased the mean clearance (CL) of ropivacaine by 31% (P<0.05), with a considerable inter-individual variation (range from -52% to +39%). It also decreased the area under the plasma concentration-time curve (AUC) of 3-OH-ropivacaine by 38% (P<0.05) and urinary excretion of 3-OH-ropivacaine by 27% (P<0.05). Ciprofloxacin increased the AUC of PPX by 71% (P<0.01) and urinary excretion of PPX by 97% (P<0.01). CONCLUSION: Ciprofloxacin modestly decreased the mean ropivacaine CL by inhibiting the CYP1A2-mediated formation of 3-OH-ropivacaine. At the same time, the CYP3A4-mediated formation of PPX was increased. There was a marked inter-individual variation in the extent of the interaction, and, for some individuals, the concomitant use of ciprofloxacin with ropivacaine might produce toxic symptoms.

Bupivacaine in the horse: relationship of local anaesthetic responses and urinary concentrations of 3-hydroxybupivacaine.

Bupivacaine is a potent local anaesthetic used in equine medicine. It is also classified as a Class 2 foreign substance by the Association of Racing Commissioners International (ARCI). The identification of residues in postrace urine samples may cause regulators to impose significant penalties. Therefore, an analytical/pharmacological database was developed for this medication. The highest no-effect dose (HNED) for the local anaesthetic effect of bupivacaine was determined to be 0.25 mg by using an abaxial sesamoid local anaesthetic model. Administration of the HNED of bupivacaine to eight horses yielded a peak urine concentration of apparent bupivacaine of 23.3 ng/mL 2 h after injection as determined with enzyme-linked immunosorbent assay (ELISA) screening. The major metabolite recovered from beta-glucuronidase-treated equine urine after dosing with bupivacaine is a hydroxybupivacaine, either 3-hydroxybupivacaine, 4-hydroxybupivacaine, or a mixture of the two. To determine which positional isomer occurs in the horse, 4-hydroxybupivacaine was obtained from Maxxam Analytics, Inc., and 3-hydroxybupivacaine was synthesized, purified, and characterized. Furthermore, a quantitative mass spectrometric method was developed for the metabolite as recovered from horse urine. Following subcutaneous injection of the HNED of bupivacaine, the concentration of the hydroxybupivacaine recovered from horse urine reached a peak of 27.4 ng/mL at 4 h after administration as measured by gas chromatography/mass spectrometry (GC/MS). It was also unequivocally demonstrated with ion chromatography that the hydroxybupivacaine metabolite found in horse urine is exclusively 3-hydroxybupivacaine and not 4-hydroxybupivacaine. The mean pH of the 4-h urine samples was 7.21; the mean urine creatinine was 209.5 mg/dL; and the mean urine specific gravity was 1.028. There was no apparent effect of pH, urine creatinine concentration, or specific gravity on the concentration of 3-hydroxybupivacaine recovered. The concentration of bupivacaine or its metabolites after administration of a HNED dose are detectable by mass spectrometric techniques. This study also suggests that recovery of concentrations less than approximately 30 ng/mL of 3-hydroxybupivacaine from postrace urine samples is unlikely to be associated with a recent local anaesthetic effect of bupivacaine.

Liquid chromatographic bioanalytical determination of ropivacaine, bupivacaine and major metabolites.

Bioanalytical methods for the determination of ropivacaine, bupivacaine and their major metabolites in urine and blood plasma are presented. Ropivacaine is a new local anaesthetic drug mainly used for surgery and for postoperative pain relief. The samples are hydrolysed and cleaned using solid-phase extraction and analysed using ion-pair reversed-phase liquid chromatography with gradient elution. The analytes are detected using UV at 210 nm. The methods are highly selective and the limits of quantification were 1 microM in urine and 0.1 microM in plasma, respectively. The between-day variance was generally below 3% (RSD).

Stereoselective urinary excretion of bupivacaine and its metabolites during epidural infusion.

A sensitive and efficient chiral assay for bupivacaine and its three principal metabolites desbutylbupivacaine, 4'-hydroxybupivacaine, and 3'-hydroxybupivacaine has been applied to urine from five male patients receiving postoperative epidural infusions of rac-bupivacaine fentanyl over 60-120 hr. The fraction of the dose of bupivacaine (total dose 840-2093 mg) accounted for in urine was 75 +/- 6%. The rate of excretion of bupivacaine enantiomers' approximated a steady state after approximately 30 hr with values of 1.27 +/- 0.26 and 0.76 +/- 0.13 mg hr-1 for (R)- and (S)-enantiomers, respectively. The fraction of the dose of bupivacaine enantiomer excreted unchanged in the urine (fe) varied from 14.3% to 39.1% for (+)-(R)-bupivacaine and 9.2% to 14.0% for (-)-(S)-bupivacaine in the five patients. The rate of excretion of all metabolites also reached a steady state after approximately 30 hr and the relative amounts of metabolites excreted into urine (fm) suggest bupivacaine is subject to regioselective and stereoselective clearance, which may vary from patient to patient.

Determination of bupivacaine and three of its metabolites in rat urine by capillary electrophoresis.

A capillary electrophoretic (CE) method for the analysis of urinary extracts of the local anesthetic, bupivacaine, and its three main metabolites, desbutylbupivacaine, 3'-hydroxybupivacaine, and 4'-hydroxybupivacaine, in rat urine has been developed. The limits of detection were 0.22 microM for desbutylbupivacaine and bupivacaine, 0.15 microM for 3'-hydroxybupivacaine, and 0.16 microM for 4'-hydroxybupivacaine. The linear range was from 0.7 microM to 16.8 microM for all four compounds. Migration time and peak height reproducibilities, and extraction efficiencies were determined for all four compounds. Peak height reproducibilities (n = 5) for the overall method were improved through the use of prilocaine as an internal standard. Peak height reproducibilities were 5.6% RSD for desbutylbupivacaine and bupivacaine, and 9.9% RSD for 3'-hydroxybupivacaine and 4'-hydroxybupivacaine. Migration time reproducibilities (n = 5) were 2.4% for all compounds. Urine samples were collected from rats administered therapeutic doses of bupivacaine and extracted using a solid-phase extraction method (SPE). Separation of bupivacaine and its metabolites was achieved in 15 min.

The application of capillary gas chromatography-selective ion mass spectrometry for the separation, identification and quantification of phenolic bupivacaine metabolites from human urine.

A capillary gas chromatographic method has been developed and validated for the separation of phenolic metabolites from human urine following the intravenous infusion of racemic bupivacaine, with subsequent formal identification and quantification by on-line mass spectrometry.

Effects of tobacco smoke exposure on the kinetics of bupivacaine in mice.

The aim of this work was to determine the effects of different time of tobacco smoke exposure on pharmacokinetics of bupivacaine in mice. Mice were exposed to tobacco smoke during 4 days (group T4) or 8 days (group T8) using the Hamburg II smoking machine. Controls were exposed under the same experimental conditions but without tobacco smoke. Serum pharmacokinetic parameters, protein or erythrocyte binding of bupivacaine were measured on the 4th and 8th day of exposure. Furthermore the urines were kept during 24 hours and urine metabolite percentages were determined. After the short exposure (4 days), no differences between treated and control groups were reported in contrary to the longer exposure (8 days), where data showed a significantly increased metabolism and elimination of bupivacaine in the treated group compared to the controls. Our data indicate that tobacco smoke acts at different levels i.e. metabolism, elimination and binding of bupivacaine. Tobacco smoke exposure increases the metabolism of bupivacaine by activating the hydroxylation route and by inducing an important elimination of 3OH-bupivacaine. Besides, it increases the permeability of the cell membranes and facilitates the penetration of bupivacaine and desbutylbupivacaine in erythrocytes.

Uptake and distribution of bupivacaine and morphine after intrathecal administration in parturients: effects of epinephrine.

This study examines the plasma pharmacokinetics of bupivacaine and morphine and the urinary excretion of morphine administered for spinal anesthesia for cesarean section. Patients received 12 mg of hyperbaric bupivacaine and 0.6 mg of morphine with either 0.2 mL of normal saline (plain, n = 15) or 0.2 mg of epinephrine (n = 15). Venous blood and urine were collected at 0, 0.25, 0.5, 1, 2, 3, 6, 12, and 24 h. Total and unconjugated morphine (UCM) in plasma and urine were measured using radioimmunoassay, and plasma bupivacaine was measured by gas chromatography. Results were expressed as mean +/- SE and analyzed using Student's t-test at P less than 0.05. In the epinephrine group, plasma bupivacaine peaked at 15 min and remained approximately 30% higher (P less than 0.05) at 0.25, 0.5, and 1 h than in the plain group, which peaked at 30 min. Although UCM peaked at 3 h in both groups, the plasma levels remained approximately 66% lower in the epinephrine group. In the epinephrine group, bupivacaine had a smaller volume of distribution at steady state, and morphine had a more rapid clearance, shorter t1/2 beta, and smaller area under the curve compared with the plain group (P less than 0.05). Only 45% +/- 5% and 31% +/- 3% of morphine was recovered in the urine in the plain and epinephrine group, respectively (P less than 0.05). In conclusion, epinephrine added to a bupivacaine-morphine mixture increases the initial systemic uptake of bupivacaine and decreases the absorption and urinary excretion of morphine from the intrathecal space.

High-performance liquid chromatographic method for the quantitation of bupivacaine, 2,6-pipecoloxylidide and 4'-hydroxybupivacaine in plasma and urine.

A high-performance liquid chromatographic method with ultraviolet detection at 210 nm for quantitation of bupivacaine and two of its metabolites from plasma and urine is described. The compounds are extracted into n-hexane-isopropanol (5:1), evaporated and the reconstituted residue injected onto a reversed phase C18 column. Standard curves for all compounds were linear (r2 greater than 0.999) in the range 20-2000 ng/ml, with a limit of detection of 10 ng/ml. The inter-day coefficients of variation ranged between 2.7 and 12.2%. The method was applied to analyse bupivacaine and metabolite concentrations in patients on long-term epidural bupivacaine-fentanyl infusions.

Effect of ranitidine on bupivacaine disposition.

This study was undertaken to determine the effect, if any, of ranitidine on bupivacaine disposition in 28 women undergoing cesarean section. Before epidural anesthesia, ranitidine (50 mg IM) or sodium citrate (30 mL orally) was administered to groups of 14 parturients each. Ranitidine was administered 2 h before epidural anesthesia and sodium citrate was administered 10 min before the epidural. Maternal plasma samples were collected after epidural anesthesia with bupivacaine. A total of 15 maternal plasma samples were taken from the time of administration of epidural anesthesia up to 180 min. Postpartum plasma and urine samples were also collected from both mothers and neonates. Plasma samples were collected up to 48 h postpartum at intervals of 12, 24, and 48 h. Urine samples were collected at six 6-h intervals up to 36 h postpartum. A two-way analysis of variance with repeated measures demonstrated that there was no significant difference in bupivacaine levels between the maternal plasma curves of the ranitidine and the control groups. At the time of delivery, plasma levels of bupivacaine and its N-dealkylated metabolite PPX (2,6-pipecolylxylidine) were no different in the mothers or neonates of either group. There was no significant difference in plasma protein binding of bupivacaine in the presence of ranitidine. The excretion rates of bupivacaine and PPX were not measurably influenced by ranitidine. The amount of bupivacaine excreted, the amount of metabolite excreted, and the percentage of drug excreted as metabolite in maternal urine were not significantly different. These data indicate that there is no measurable effect of ranitidine on the disposition of bupivacaine in parturients.

Simultaneous determination of bupivacaine and its two metabolites, desbutyl- and 4'-hydroxybupivacaine, in human serum and urine.

A sensitive and selective high-performance liquid chromatographic (HPLC) method for the determination of bupivacaine and its two metabolites, desbutyl- and 4'-hydroxybupivacaine, in human serum and urine is described. Bupivacaine, both metabolites and the internal standard, etidocaine, are extracted with diethyl ether and then back-extracted into an acidic aqueous phase. After subsequent extraction into diethyl ether, evaporation and reconstitution in the mobile phase, bupivacaine and the metabolites are determined by HPLC using a reversed-phase C8 column with tetrahydrofuran-potassium phosphate buffer (8:92, v/v, pH 2.4) as the mobile phase. The sensitivity of the method is 10 micrograms/l for bupivacaine and both metabolites and the extraction efficiencies are 95, 54 and 92% for bupivacaine and desbutyl- and 4'-hydroxybupivacaine, respectively. The reproducibility of the method is good, the coefficients of variation varying between 1.8 and 7.4% in the concentration range 0.10-2.00 mg/l. The procedure was applied to human serum and urine samples from two elderly women who had been operated on under epidural analgesia (plain bupivacaine, 1.5 mg/kg) because of uterine prolapse.