Stereospecific analysis of sakuranetin by high-performance liquid chromatography: pharmacokinetic and botanical applications

A stereospecific method for analysis of sakuranetin was developed. Separation was accomplished using a Chiralpak AD-RH column with UV (ultraviolet) detection at 288 nm. The stereospecific linear calibration curves ranged from 0.5 to 100 microg/mL. The mean extraction efficiency was >98%. Precision of the assay was <12% (relative standard deviation (R.S.D.)%), and within 10% at the limit of quantitation (0.5 microg/mL). Bias of the assay was lower than 10%, and within 5% at the limit of quantitation. The assay was applied successfully to pharmacokinetic quantification in rats, and the stereospecific quantification in oranges, grapefruit juice, and matico (Piper aduncum L.).     

Stereospecific high-performance liquid chromatographic analysis of tramadol and its O-demethylated (M1) and N,O-demethylated (M5) metabolites in human plasma

A stereospecific method for simultaneous quantitation of the enantiomers of tramadol (T) and its active metabolites O-demethyl tramadol (M1) and O-demethyl-N-demethyl tramadol (M5) in human plasma is reported. After the addition of penbutolol (IS), plasma (0.5 ml) samples were extracted into methyl tert-butyl ether, followed by back extraction into an acidic solution. The separation was achieved using a Chiralpak AD column with a mobile phase of hexanes:ethanol:diethylamine (94:6:0.2) and a flow rate of 1 ml/min. The fluorescence of analytes was then detected at excitation and emission wavelengths of 275 and 300 nm, respectively. All the six enantiomeric peaks of interest plus three unknown metabolite peaks and IS peak (a total of 10 peaks) eluted within 23 min, free from endogenous interference. The assay was validated in the plasma concentration range of 2.5-250 ng/ml, with a lower limit of quantitation of 2.5 ng/ml, for all the six analytes. The extraction efficiency (n=5) was close to 100% for both T and M1 enantiomers and 85% for M5 and IS enantiomers. The application of the assay was demonstrated by simultaneous measurement of plasma concentrations of T, M1, and M5 enantiomers in a healthy volunteer after the administration of 50 mg oral doses of racemic T.     

Stereospecific high-performance liquid chromatographic analysis of ibuprofen in rat serum

A simple, rapid and sensitive high-performance liquid chromatographic method was developed for determination of ibuprofen, (+/-)-(R, S)-2-(4-isobutylphenyl)-propionic acid, enantiomers in rat serum. Serum (0.1 ml) was extracted with 2,2,4-trimethylpentane/isopropanol (95:5, v/v) after addition of the internal standard, (S)-naproxen, and acidification with H(2)SO(4). Enantiomeric resolution of ibuprofen was achieved on ChiralPak AD-RH column with ultraviolet (UV) detection at 220 nm without interference from endogenous co-extracted solutes. The calibration curve demonstrated excellent linearity between 0.1 and 50 microg/ml for each enantiomer. The mean extraction efficiency was >92%. Precision of the assay was within 11% (relative standard deviation (R.S.D.)) and bias of the assay was lower than 15% at the limit of quantitation (0.1 microg/ml). The assay was applied successfully to an oral pharmacokinetic study of ibuprofen in rats.     

Enantioselective analysis of glufosinate using precolumn derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate and reversed-phase liquid chromatography

We have developed a new analytical method to quantify the DL-homoalanine-4-yl(methyl)phosphinate (DL-GLUF) enantiomers in biological specimens using a reversed-phase high-performance liquid chromatography system with a fluorescence detection system. The derivatization of DL-GLUF enantiomers with (+)-1-(9-fluorenyl)ethyl chloroformate was carried out under mild conditions (40 degrees C for 30 min) without inducing racemization. The lower limit of quantitation was 0.01 microg/ml for both D-GLUF and L-GLUF, and the detection limit was 5 ng/ml. When DL-GLUF enantiomers were added to serum to produce concentrations between 0.1 and 100 microg/ml, the mean recovery rate was at least 93.8%. The recovery rate from urine was also satisfactory.     

Quantitation of trimipramine enantiomers in human serum by enantioselective high-performance liquid chromatography and mixed-mode disc solid-phase extraction

A sensitive and stereospecific method for the quantitation of trimipramine enantiomers in human serum was developed. The assay involves the use of a novel mixed-mode disc solid-phase extraction for serum sample clean-up prior to HPLC analysis and is also free of interference from the enantiomers of desmethyltrimipramine, 2-hydroxytrimipramine, and 2-hydroxydesmethyltrimipramine, the three major metabolites of trimipramine. Chromatographic resolution of trimipramine enantiomers was performed on a reversed-phase cellulose-based chiral column (Chiralcel OD-R) under isocratic conditions using a mobile phase consisting of 0.3 M aqueous sodium perchlorate-acetonitrile (58:42, v/v) at a flow-rate of 0.5 ml/min. Recoveries for R- and S-trimipramine enantiomers were in the range of 93–96% at 25–185 ng/ml levels. Intra-day and inter-day precisions calculated as R.S.D. were in the ranges of 0.30-8.00% and 1.60-10.20% for both enantiomers, respectively. Intra-day and inter-day accuracies calculated as percent error were in the 0.01–2.10% and 1.00–3.00% ranges for both enantiomers, respectively. Linear calibration curves were in the concentration range 15–250 ng/ml for each enantiomer in serum. The limit of quantification of each enantiomer was 15 ng/ml. The detection limit for each enantiomer in serum using a UV detector set at 210 nm was 10 ng/ml (S/N =2). In addition, separation of the enantiomers of desmethyltrimipramine, 2-hydroxytrimipramine, and 2-hydroxydesmethyltrimipramine were investigated. The desmethyltrimipramine enantiomers could be resolved on the Chiralcel OD-R column under the same chromatographic conditions as the trimipramine enantiomers, but the other two metabolite enantiomers required different mobile phases on the Chiralcel OD-R column to achieve satisfactory resolution with R_s values of 1.00.     

A sensitive gas chromatographic/mass spectrometric method for the resolution and quantification of ethosuximide enantiomers in biological fluids

A modified specific, sensitive and reproducible chiral gas chromatographic (GC) method for the resolution and quantification of ethosuximide enantiomers in urine and plasma was developed. The samples were extracted by liquid-liquid extraction, using diethylether and the enantiomers were separated and quantified on a chiral gas chromatographic column (25QC2 / CYDEX- beta 0.25). The method involved the use of GC/MS instrumentation for the acquisition of data in the electron impact selective-ion monitoring mode, collecting ions characteristic of both ethosuximide and alpha, alpha - dimethyl - beta - methylsuccinimide, the internal standard and of mass-to-charge ratio (m/z) exactly equal to 55 and 70 units. The limit of quantitation of the method was 2.5 microg/ml for both urine and plasma with both enantiomers. The method proved to be linear, precise and reproducible in the 5-300 microg/ml concentration range for urine samples and in the 10-250 microg/ml concentration range for plasma samples. Future research work envisaged the application of this method in pharmacokinetic and pharmacodynamic studies.     

Determination of disodium clodronate in human plasma and urine using gas-chromatography-nitrogen-phosphorous detections: validation and application in pharmacokinetic study

We present a specific method for the determination of disodium clodronate in human plasma and urine using a gas-chromatographic system with nitrogen phosphorus detector (NPD). The compound was extracted from plasma and urine samples by an anion-exchange resin and derivatizated with bistrimethylsilyltrifluoroacetamide (BSTFA). Sodium bromobisphosphonate was used as internal standard. The calibration curves were linear in both plasma and urine, with a regression coefficient r > 0.9975 in plasma and r > 0.9977 in urine.The limit of quantitation was 0.3 microg/ml in plasma and 0.5 microg/ml in urine. The method was validated by intra-day assays at three concentration levels. During the study we carried out inter-day assays to confirm the feasibility of the method. The precision in plasma at 0.5, 15, and 45 microg/ml was 12.4, 0.2, and 6.5% (n = 40), respectively; in urine at 0.8, 8, and 40 microg/ml it was 8.6, 6.4, and 9.3% (n = 40), respectively.The method was accurate and reproducible, and was successfully applied to determine the pharmacokinetic parameters of clodronate in healthy volunteers after intravenous infusion and intramuscular injection of 200 mg of the compound. The Cmax after intravenous infusion and intramuscular injection was 16.1 and 12.8 microg/ml, respectively. AUC(0-48 h) after infusion administration and intramuscular injection was 44.2 +/- 18.0 and 47.5 +/- 12.4 h microg/ml, respectively. The elimination half-life in both administrations was 6.31 +/- 2.7 h.     

Determination of L-threonate in human plasma and urine by high performance liquid chromatography-tandem mass spectrometry

A fast and selective HPLC-MS-MS method was established to determine L-threonate in human plasma and urine. Plasma and urine samples were extracted by protein precipitation and diluted with water, then chromatographed on an YMC J'Sphere C(18) column with methanol-acetonitrile-10mM ammonium acetate (20:5:75, v/v) as mobile phase, and at a flow rate of 0.2 ml/min. Detection was performed on a triple-quadrupole tandem mass spectrometer using negative electrospray ionization (ESI). Multiple reactions monitoring (MRM) was used and L-threonate was quantified by monitoring the ion transition of m/z 134.5-->74.7. The linear calibration curves of L-threonate in plasma and urine were obtained over the concentration range of 0.25-50 microg/ml and 2.5-500 microg/ml, respectively. Lower limit of quantitation was 0.25 and 2.5 microg/ml, respectively. Accuracy was within 85-115%, and intra- and inter-batch precision (R.S.D.%) were within +/-15%. The method proved to be accurate and specific, and was applied to the pharmacokinetic study of L-threonate in Chinese healthy subjects.     

Determination of gacyclidine enantiomers in human plasma by gas chromatography–mass spectrometry using selected-ion monitoring

A sensitive gas chromatographic assay using mass selective-detection has been developed for the simultaneous quantitation of the enantiomers of (±)-gacyclidine (a non competitive N-methyl-

Full-size image

-aspartate antagonist) in human plasma. Gacyclidine enantiomers and phencyclidine (PCP), the internal standard, were extracted using a single-step liquid–liquid extraction with hexane at pH 8.0. Each enantiomer was separated on a chiral gas chromatography capillary column and specifically detected by mass spectrometry (MS) in selected-ion monitoring (SIM) mode. Gacyclidine enantiomers and PCP were monitored using the fragment ions at m/z 206 and 200, respectively. No interference was observed from endogenous components. The limit of quantitation (LOQ) for each enantiomer of gacyclidine was 300 pg/ml by using plasma samples of 500 μl. The calibration curves were linear (r²=0.998) over a range of 0.3125 to 20 ng/ml. The extraction efficiency was higher than 95% for both enantiomers. Intra- and inter-day bias were less than 10% at every standard curve concentration. Intra-day precision was less than 19% for (−)-gacyclidine and 15% for (+)-gacyclidine. Inter-day precision was below 15% for both enantiomers. The assay was validated for an enantioselective pharmacokinetic study in healthy male volunteers.     

Column-switching technique for the sensitive determination of ertapenem in human cerebrospinal fluid using liquid chromatography and ultraviolet absorbance detection

A sensitive reversed-phase high-performance liquid chromatographic (RP-HPLC) assay with on-line extraction was developed for quantifying ertapenem in human cerebrospinal fluid (CSF). This assay is at least five times more sensitive than previously published ertapenem methods with a lower limit of quantitation at 0.025 microg/ml. In this assay, a CSF sample is extracted on-line using a RP extraction column and an aqueous acidic mobile phase (0.1% formic acid) to wash away polar endogenous materials, while ertapenem is retained on the column. Ertapenem is then back-flushed off the extraction column and directed to a RP analytical column using an acidic mobile phase with an organic modifier (acetonitrile/0.1% formic acid, 15:85 (v/v)) and detected using UV absorbance. The acidic mobile phase provided a sharper chromatographic peak and on-line extraction allowed large injection volumes (> or = 150 microl) of buffered CSF to be injected without compromising column integrity. These assay conditions were necessary to quantify ertapenem at levels expected to be found in human CSF (< 0.05 microg/ml). The method was successfully validated and implemented for a clinical study: intraday precision and accuracy of the CSF assay for calibration standards (0.025-10 microg/ml) and quality control samples (0.1, 0.5, and 2.5 microg/ml) were < 6.2% coefficient of variation and 96.8-104.0% of nominal concentration, respectively.     

Enantioselective determination of oxprenolol and its metabolites in human urine by cyclodextrin-modified capillary zone electrophoresis

A stereospecific capillary electrophoresis assay for oxprenolol enantiomers and their basic metabolites in human urine has been developed using hydroxypropyl-β-CD as a chiral selector in the mobile phase. The bioassay method has been validated and the detection limit from spiked urine samples is 0.2μg/ml. The calibration curves are linear from 0.4 to 16 μg/ml. Extraction recovery ranged from 84.7 to 96.4% for all the compounds studied. The influence of various parameters on the chiral separation of oxprenolol and its basic metabolites have been investigated. Urinary excretion profiles of oxprenolol enantiomers and those of two metabolites have also been studied, following a single oral dose of racemic oxprenolol.     

Enantioselective pharmacokinetics of homochlorcyclizine: III. Simultaneous determination of (+)- and (−)- homochlorcyclizine in human urine by high-performance liquid chromatography

A method is described for the simultaneous determination of (+)- and (−)-homochlorcyclizine (HCZ) in human urine by high-performance liquid chromatography on a chiral stationary phase of ovomucoid-bonded silica. The pH of the buffer and organic modifier in the mobile phase markedly affected the chromatographic separation. A mobile phase of methanol—0.02 M acetate buffer (pH 4.7) (25:75, v/v) at a flow-rate of 1.0 ml/min was used for the urine assays. The ultraviolet absorption was monitored at 240 nm, and diphenhydramine was employed as the internal standard for the quantitation. (+)-HCZ, (−)-HCZ and the internal standard were eluted at retention times of 15, 25 and 8 min, respectively. The limit of determination for HCZ enantiomers was ca. 50 ng/ml of urine. One of the metabolites in human urine, which was a quaternary ammonium-linked glucuronide, could also be determined in a manner similar to unchanged HCZ after β-glucuronidase hydrolysis. A pharmacokinetic study was conducted with three healthy volunteers, who each received a single oral dose of racemic HCZ (20 mg). Distinct differences were found between the two enantiomers, particularly in the metabolic process, that is, the urinary excretion as (−)-HCZ-glucuronide within 48 h was ca. four times higher than that of the (+)-isomer. This method should be very useful for enantioselective pharmacokinetic studies of HCZ.     

Simultaneous determination of the enantiomers of esmolol and its acid metabolite in human plasma by reversed phase liquid chromatography with solid-phase extraction

A stereoselective RP-high performance liquid chromatography (HPLC) assay to determine simultaneously the enantiomers of esmolol and its acid metabolite in human plasma was developed. The method involved a solid-phase extraction and a reversed-phase chromatographic separation with UV detection (lambda = 224 nm) after chiral derivatization. 2,3,4,6-tetra-O-acetyl-beta-d-glucopyranosyl isothiocyanate (GITC) was employed as a pre-column chiral derivatization reagent. The assay was linear from 0.09 to 8.0 microg/ml for each enantiomer of esmolol and 0.07-8.0 microg/ml for each enantiomer of the acid metabolite. The absolute recoveries for all enantiomers were >73%. The intra- and inter-day variations were <15%. The validated method was applied to quantify the enantiomers of esmolol and its metabolite in human plasma for hydrolysis studies.     

Enantioselective determination of FCE 28833, a new potential antiischemic agent,in gerbil plasma using column switching HPLC with UV detection

A sensitive and selective high performance liquid chromatographic method using an automated column switching technique for the determination of FCE 28833 enantiomers in gerbil plasma was developed. After solid-liquid extraction using a Supelcosil C₁₈ cartridge FCE 28833 was eluted on a clean-up column (Spherisorb CN) and the enantiomers were separated using an analytical chiral column (Crownpack CR(+)). The mobile phase (15% methanol in HClO₄ 1 mM) was directed through the columns at a flow rate of 1 ml/min and the fraction eluted between 13 and 40 min was transferred from the clean-up column into the analytical column. FCE 28833 enantiomers were monitored at 257 nm. The limit of quantitation of the method was 20 ng/ml plasma for both enantiomers and proved to be linear, precise, and accurate for the assay of both enantiomers in the 20–6,000 ng/ml concentration range. No interference from the blank gerbil plasma sample was observed. The suitability of the method was assessed using plasma samples obtained from male gerbils treated with a single oral dose (400 mg/kg) of FCE 28833. Chirality 9:133–138, 1997. © 1997 Wiley-Liss, Inc.     

Determination of amdinocillin in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of amdinocillin (formerly mecillinam) in human plasma and urine. The assay is performed by direct injection of a plasma protein-free supernatant or a dilution of urine. A 10-μm μBondapak phenyl column with an eluting solvent of water—methanol—1 M phosphate buffer, pH 7 (70:30:0.5) was used, with UV detection of the effluent at 220 nm. Azidocillin potassium salt [potassium-6-(d-(-)-α-azidophenyacetamido)-penicillanate] was used as the internal standard and quantitation was based on peak height ratio of amdinocillin to that of the internal standard. The assay has a recovery of 74.4 ± 6.3% (S.D.) in the concentration ranges of 0.1–20 μg per 0.2 ml of plasma with a limit of detection equivalent to 0.5 μg/ml plasma. The urine assay was validated over a concentration range of 0.025–5 mg/ml of urine, and has a limit of detection of 0.025 mg/ml (25 μg/ml) using a 0.1-ml urine specimen per assay.The assay was applied to the determination of plasma and urine concentrations of amdinocillin following intravenous administration of a 10 mg/kg dose of amdinocillin to two human subjects. The HPLC and microbiological assays were shown to correlate well for these samples.     

Sensitive determination of bisoprolol enantiomers in plasma and urine by high-performance liquid chromatography using fluorescence detection, and application to preliminary study in humans

A sensitive, stereoselective high-performance liquid chromatographic method with fluorescence detection for the measurement of bisoprolol enantiomers in human plasma and urine has been developed. Bisoprolol was extracted at alkaline pH with chloroform, followed by solid-phase extraction. The effluent was evaporated, and the reconstituted residue was chromatographed on a Chiralcel OD column with a mobile phase of hexane—2-propanol (10:0.9, v/v) containing 0.01% (v/v) diethylamine. Within the plasma and urine enantiomeric concentration ranges of 5–100 ng/ml and 25–1250 ng/ml, respectively, a linear relationship was obtained between the peak-height ratios and the corresponding concentrations. The limit of quantitation, defined as three times the baseline noise, was 2 ng/ml for each enantiomer in plasma. A preliminary pharmacokinetic study was undertaken in three healthy male volunteers following an oral dose of 5 mg of racemic bisoprolol. The results confirm that this assay is suitable for pharmacokinetic studies of bisoprolol enantiomers in humans following oral administration of the therapeutic dose.     

Liquid chromatography-tandem mass spectroscopy assay for quercetin and conjugated quercetin metabolites in human plasma and urine

A sensitive and specific method was developed and validated for the quantitation of quercetin in human plasma and urine. The application of liquid chromatography-tandem mass spectrometry (LC/MS/MS) with a TurboIonspray (TIS) interface in negative mode under multiple reactions monitoring was investigated. Chromatographic separation was achieved on a C12 column using a mobile phase of acetonitrile/water with 0.2% formic acid (pH 2.4) (40/60, v/v). The detection limit was 100 pg/ml and the lower limit of quantification was 500 pg/ml for plasma samples; the detection limit was 500 pg/ml and the lower limit of quantification was 1 ng/ml for urine samples. The calibration curve was linear from 1 to 800 ng/ml for plasma samples and was linear from 1 to 200 and 50 to 2000 ng/ml for urine samples. All the intra- and inter-day coefficients of variation were less than 11% and intra- and inter-day accuracies were within +/-15% of the known concentrations. This represents a LC/MS/MS assay with the sensitivity and specificity necessary to determine quercetin in human plasma and urine. This assay was used to determine both parent quercetin and the quercetin after enzymatic hydrolysis with beta-glucuronidase/sulfatase in human plasma and urine samples following the ingestion of quercetin 500 mg capsules.     

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.     

High-performance liquid chromatographic analysis of methocarbamol enantiomers in biological fluids

Methocarbamol enantiomers in rat and human plasma were quantified using a stereospecific high-performance liquid chromatographic method. Racemic methocarbamol and internal standard, (R)-(−)-flecainide, were isolated from plasma by a single-step extraction with ethyl acetate. After derivatization with the enantiomerically pure reagent (S)-(+)-1-(1-naphthyl)ethyl isocyanate, methocarbamol diastereomers and the (R)-flecainide derivative were separated on a normal-phase silica column with a mobile phase consisting of hexane—isopropanol (95:5, v/v) at a flow-rate of 1.6 ml/min. Ultraviolet detection was carried out at a wavelength of 280 nm. The resolution factor between the diastereomers was 2.1 (α = 1.24). An excellent linearity was observed between the methocarbamol diastereomers/internal standard derivative peak-area ratios and plasma concentrations, and the intra- and inter-day coefficients of variation were always <9.8%. The lowest quantifiable concentration was 0.5 μg/ml for each enantiomer (coefficients of variation of 9.8 and 8.8% for (S)- and (R)-methocarbamol, respectively), while the limit of detection (signal-to-noise ratio 3:1) was approximately 10 ng/ml. The assay was used to study the pharmacokinetics of methocarbamol enantiomers in a rat following intravenous administration of a 120 mg/kg dose of racemic methocarbamol and to evaluate plasma and urine concentrations in a human volunteer after oral administration of a 1000-mg dose of the racemate. The method is suitable for stereoselective pharmacokinetic studies in humans as well as in animal models.     

Development and validation of HPLC method for the determination of tobramycin in urine samples post-inhalation using pre-column derivatisation with fluorescein isothiocyanate

A reversed-phase liquid chromatography method involving pre-column derivatisation with fluorescein isothiocyanate (FITC, isomer I) for determination of tobramycin in urine samples after inhalation has been developed. FITC reacts with the primary amino groups of tobramycin and other aminoglycosides under mild conditions to form a highly fluorescent and stable derivative. The chromatographic separation was carried out on a Phenomenex Luna C(18) column at ambient temperature using a constant flow rate of 1 ml/min and mobile phase of acetonitrile-methanol-glacial acetic acid-water (420:60:5:515, v/v/v/v). The tobramycin-FITC derivative was monitored by fluorescent detection at an excitation wavelength 490 nm and emission wavelength 518 nm. The linearity of response for tobramycin was demonstrated at 11 different concentrations of tobramycin extracted from spiked urine, ranging from 0.25 to 20 microg/ml. Tobramycin and neomycin were extracted from spiked urine by a solid phase extraction clean-up procedure on a carboxypropyl-bonded phase (CBA) weak cation-exchange cartridge, and the relative recovery was >99% (n=5). The limit of detection (LOD) and limit of quantitation (LOQ) in urine were 70 and 250 ng/ml, respectively. The method had an accuracy of <0.2%, and intra-day and inter-day precision (in term of %coefficient of variation) were <4.89% and 8.25%, respectively. This assay was used for urinary pharmacokinetic studies to identify the relative lung deposition of tobramycin post-inhalation of tobramycin inhaled solution 300 mg/5 ml (TOBI) by different nebuliser systems.