Journal of Chromatography B (v.785, #1)
Editorial Board (iii).
News Section (N1-N2).
Chromatographic performance of large-pore versus small-pore columns in micellar liquid chromatography by Timothy J McCormick; Joe P Foley; David K Lloyd (1-20).
Micellar liquid chromatography (MLC) is useful in bioanalysis because proteinaceous biofluids can be directly injected onto the column. The technique has been limited in part because of the apparently weak eluting power of micellar mobile phases. It has recently been shown [Anal. Chem. 72 (2000) 294] that this may be overcome by the use of large pore size stationary phases. In this work, large-pore (1000 Å) C18 stationary phases were evaluated relative to conventional small-pore (100 Å) C18 stationary phases for the direct sample injection of drugs in plasma. Furthermore, the difference between the large and small pore phases in gradient elution separations of mixtures of widely varying hydrophobicities was investigated. Large-pore stationary phases were found to be very effective for eluting moderately to highly hydrophobic compounds such as ibuprofen, crotamiton, propranolol, and dodecanophenone, which were highly retained on the small-pore stationary phases typically used in MLC. The advantages of direct introduction of biological samples (drugs in plasma) and rapid column re-equilibration after gradient elution in MLC were maintained with large-pore phases. Finally, recoveries, precision, linearity, and detection limits for the determination of quinidine and DPC 961 in spiked bovine plasma were somewhat better using MLC with wide pore phases.
Keywords: Highly retained compounds;
Alkylation of DNA by melphalan: investigation of capillary liquid chromatography–electrospray ionization tandem mass spectrometry in the study of the adducts at the nucleoside level by Bart Van den Driessche; Filip Lemière; Walter Van Dongen; Eddy L Esmans (21-37).
Nitrogen mustards are among the oldest cancer chemotherapeutic agents and remain the drugs of choice for treatment of many human cancers. A serious complication of treatment with nitrogen mustards is the increased risk of a secondary leukaemia in long-term survivors because not all alkylating agent interactions with DNA result in cell death. In an earlier study 2′-deoxy-5′-mononucleotide/melphalan adducts have been analysed by us by LC–ES MSMS. In this work we want to present the first results of the analysis of the corresponding 2′-deoxynucleoside/melphalan adducts from DNA hydrolysates by column switching/capillary LC–ES tandem mass spectrometry. Nucleosides, compared to nucleotides, give better chromatographic results and show a good sensitivity under electrospray (+) [ES(+)] ionisation. Several adducts were identified under ES(+) conditions. Mono-alkylated nucleoside adducts alkylated at the base moiety were identified for dGuo, dCyd and dAdo. Structures were identified by recording the low-energy CAD product ion scans. Also a mono-alkylated nucleotide pdA with alkylation position at the phosphate moiety could be detected. This proves that in the case of phosphate alkylation the enzymatic dephosphorylation reaction was inhibited. A Jurkat cell suspension was treated with melphalan (1 mM) and incubated at 37 °C (5% CO2). After 6 and 48 h, the DNA was isolated and enzymatically hydrolysed. The corresponding nucleoside pool was evaluated with the developed LC–MS method. In the 48-h experiment, one adduct could be identified as a N-7 alkylated dGuo. In the 6-h experiment, no adducts could be found. Additional experiments were done wherein Jurkat-DNA, isolated from a non-treated cell culture, was treated with melphalan. These results were analogous with the data found in melphalan-treated calf thymus DNA. Additionally, we tried to determine the exact alkylation position by interpreting high-resolution fragmentation spectra.
Keywords: DNA; Melphalan; Nucleosides;
Determination of riboflavin in urine by capillary electrophoresis–blue light emitting diode-induced fluorescence detection combined with a stacking technique by An-Kai Su; Cheng-Huang Lin (39-46).
A simple, inexpensive and reliable method for the simultaneous, routine analysis of riboflavin in urine by capillary electrophoresis–light emitting diode (LED)-induced fluorescence detection is described. Using a blue LED as the light source, the detection limit of riboflavin was determined to be 0.48 μg/ml and was improved to 20 ng/ml when a stacking technique was applied. In the analysis of an actual sample, various concentrations of riboflavin were distributed in the urine samples over a period of 9 h after the ingestion of a vitamin B2 tablet.
Liquid chromatography–tandem mass spectrometry analysis of oleuropein and its metabolite hydroxytyrosol in rat plasma and urine after oral administration by Piero Del Boccio; Antonietta Di Deo; Amalia De Curtis; Nicola Celli; Licia Iacoviello; Domenico Rotilio (47-56).
We describe a liquid chromatography–electrospray ionisation tandem mass spectrometry method for the qualitative and quantitative determination of the secoiridoid oleuropein and its bioactive metabolite hydroxytyrosol in rat plasma and urine. Samples were prepared by liquid–liquid extraction using ethyl acetate with a recovery for both compounds of about 100% in plasma and about 60% in urine. The chromatographic separation was performed with a RP-ODS column using a water–acetonitrile linear gradient. The calibration curve was linear for both biophenols over the range 2.5–1000 ng/ml (LOD 1.25 ng/ml) for plasma and 5–1000 ng/ml (LOD 2.5 ng/ml) for urine. Plasma concentrations of oleuropein and hydroxytyrosol were measured after oral administration of a single dose (100 mg/kg) of oleuropein. Analysis of treated rat plasma showed the presence of unmodified oleuropein, reaching a peak value of 200 ng/ml within 2 h, with a small amount of hydroxytyrosol, whereas in urine, both compounds were mainly found as glucuronides.
Keywords: Oleuropein; Hydroxytyrosol;
Determination of uric acid in human saliva by high-performance liquid chromatography with amperometric electrochemical detection by Koichi Inoue; Tatsuya Namiki; Yusuke Iwasaki; Yoshihiro Yoshimura; Hiroyuki Nakazawa (57-63).
The aim of the present study is to establish a highly sensitive method for the determination of uric acid (UA) in human saliva. The monitoring of UA levels in less invasive biological samples such as saliva is suggested for the diagnosis and therapy of gout, hyperuricemia, and the Lesch–Nyhan syndrome, and for detecting such conditions as alcohol dependence, obesity, diabetes, high cholesterol, high blood pressure, kidney disease, and heart disease. Reversed-phase high-performance liquid chromatography with electrochemical detection (HPLC–ED) was employed for the determination of UA obtained by solid-phase extraction from saliva. To quantify UA, we compared the ED efficiencies of an amperometric ED (Ampero-ED) with a single electrode and a coulometric ED (Coulo-ED) with a multiple electrode array. The results showed that the detection limits (S/N=3) were 3 nM for Ampero-ED and 6 nM for Coulo-ED, and the linearity of the calibration curves of 60–6000 nM had correlation coefficients exceeding 0.999. In addition, the total analytical time was 10 min. In the sample preparation of UA in saliva, an Oasis MAX solid-phase cartridge was used. The recoveries of UA spiked at 0.6 and 3 μM in saliva were above 95% with a relative standard deviation (RSD) of less than 15%. Therefore, the present method may be used in the routine and diagnostic determination of UA in human saliva.
Keywords: Uric acid;
Measurement of the anti-cancer agent gemcitabine in human plasma by high-performance liquid chromatography by Bruce Keith; Yan Xu; Jean L. Grem (65-72).
A reversed-phase HPLC assay has been developed to determine the concentration of the anti-metabolite 2′,2′-difluorodeoxycytidine (gemcitabine, dFdC) in human plasma over the concentration range of 0.5–150 μM (0.13–39.44 μg/ml), and 2′,2′-difluorodeoxyuridine (dFdU), the deaminated, inactive metabolite, over the range of 1.0–227 μM (0.26–60 μg/ml). After the addition of 20 nmol 2′-fluorodeoxycytidine (FdC) as an internal standard, 0.5-ml samples of plasma were subjected to acetonitrile precipitation, followed by analysis using a gradient reversed-phase HPLC assay with UV detection. A Phenomenex Columbus™ C18 column, 5 μm, 150×4.6 mm, and a Waters C18, 4 μm, Nova-Pak Sentry guard column were used to achieve separation. FdC, dFdC and dFdU were monitored at 282, 269 and 258 nm, respectively, on a Waters 996 photodiode array detector. The mobile phase, run at a total flow-rate of 1.5 ml/min, was composed of two solvents: 50 mM ammonium acetate pH 5.0 in either 2% (solvent A) or 10% methanol (solvent B, v/v); 100% solvent A was run for 17 min, followed by a linear gradient to 100% solvent B over 14 min. FdC, dFdC and dFdU were resolved from endogenous compounds and had retention times of 13.6±0.5, 18.1±1.1 and 29.0±0.6 min, respectively. The assay was useful in measuring the plasma levels of both analytes in samples obtained from adult cancer patients participating in a Phase I trial of gemcitabine given as either a 1- or 2-h infusion weekly for 3 of 4 weeks.
Validation of a simple gas chromatographic–mass spectrometric method for the determination of gamma-butyrolactone in human plasma by Yousuke Fukui; Eiji Matsusima; Kouichi Muramoto; Nobutaka Nagai; Koso Ohama; Kazumasa Yamashita (73-80).
A gas chromatographic–mass spectrometric (GC–MS) method is described for the determination of human plasma levels of gamma-butyrolactone (GBL) is described. The method is sensitive and simple. The plasma sample spiked with the internal standard was extracted by dichloromethane (CH2Cl2) in acidic conditions, and the concentrated organic layer was injected into GC–MS. Because of endogenous GBL in human plasma, the method used a standard calibration curve. The calibration curve was linear from 10 to 1000 ng/ml. The method has been validated for accuracy and precision with the relative error and C.V. for intra- and inter-day within 10%. GBL-spiked plasma samples stored at −80 °C were stable for a 3-month period. The stability of plasma samples after three cycles of freezing and thawing and of prepared samples on an autosampler for 48 h were demonstrated. Plasma concentrations of GBL before and after administration of UFT were 24.3±14.2 and 84.9±22.4 ng/ml, respectively.
Quantification of the O- and N-demethylated metabolites of hydrocodone and oxycodone in human liver microsomes using liquid chromatography with ultraviolet absorbance detection by Andrew Menelaou; Mark R. Hutchinson; Ingvild Quinn; Anders Christensen; Andrew A. Somogyi (81-88).
High-performance liquid chromatographic assays for the O- and N-demethylated oxidative metabolites of hydrocodone and oxycodone formed in human liver microsomes are described. A solvent–solvent extraction/re-extraction procedure followed by reversed-phase HPLC with UV detection at 210 nm allows for the quantification of hydromorphone, norhydrocodone, oxymorphone and noroxycodone. Calibration curve concentration ranges were 0.63–400 μM (0.18–114 μg/ml) and 1.25–400 μM (0.36–114 μg/ml) for hydromorphone and norhydrocodone, respectively and 0.13–20 μM (0.04–6.03 μg/ml) and 1–200 μM (0.30–60 μg/ml) for oxymorphone and noroxycodone, respectively. Assay performance was determined by intra- and inter-assay precision and inaccuracies for quality control samples and was <15% for all metabolites at each quality control concentration. These methods provide good precision, accuracy and sensitivity for use in in vitro kinetic studies investigating the oxidative metabolism of hydrocodone and oxycodone in human liver microsomes.
Keywords: Norhydrocodone; Hydromorphone; Noroxycodone; Oxymorphone;
Simultaneous determination of molecular species of monoacylglycerols, diacylglycerols and triacylglycerols in human very-low-density lipoproteins by reversed-phase liquid chromatography by Javier S. Perona; Valentina Ruiz-Gutierrez (89-99).
The aim of the present study was to investigate the applicability of a previously developed method for the analysis of triacylglycerol molecular species to the simultaneous determination of triacylglycerols, diacylglycerols and monoacylglycerols of human very-low-density lipoproteins (VLDL). Ten elderly women were recruited for the study. Blood was obtained in fasting conditions and VLDL were isolated by ultracentrifugation. Neutral lipids were separated by solid-phase extraction and were subsequently injected on a reversed-phase HPLC system, with an elution system composed of acetone in acetonitrile. The method allowed the separation of four monoacylglycerols, 18 diacylglycerols and 24 triacylglycerols, including the resolution of positional isomers of diacylglycerols. Monoacylglycerols were composed of oleic, linoleic, palmitic and stearic acids. The major diacylglycerols were 1,2-dilinoleoyl-glycerol and 1,3-dilinoleoyl-glycerol (14.24±1.02 and 17.93±1.42%, respectively). The main triacylglycerols quantified were dioleoyl-stearoyl-glycerol (OOS), oleoyl-dipalmitoyl-glycerol (OPP), trilinoleoyl-glycerol (LLL) and linoleoyl-distearoyl-glycerol (LSS), accounting for 11.25±2.15, 10.14±2.05, 9.35±2.30 and 8.56±1.56%, respectively. An inverse relationship between polarity and fatty acid disappearance from triacylglycerols (r 2=0.82, P<0.05) and from diacylglycerols (r 2=0.93, P<0.01) was discovered. In conclusion, the method allowed, for the first time, the easy, rapid and simultaneous determination in a single chromatogram of triacylglycerol, diacylglycerol and monoacylglycerol molecular species of human VLDL by reversed-phase HPLC.
Keywords: Acylglycerols; Very-low-density lipoproteins;
Improved method for measurement of human plasma xanthine oxidoreductase activity by X Liu; W.M Lin; X.H Yan; X.H Chen; J.R Hoidal; P Xu (101-114).
The XOR activity in human plasma was measured by quantifying the XOR-derived uric acid (UA) in plasma using the high-performance liquid chromatography (HPLC) equipped with a UV detector. Chromatographic separation consisted of the mobile phase (a mixture of 0.1% trifluoroacetic acid in Milli-Q water and 0.085% trifluoroacetic acid in acetonitrile in a mix ratio of 99:1) running through a Zorbax StableBond SB-C18 column at a flow-rate of 1 ml/min. Deproteinization with heat-treatment of plasma samples after the reaction was used in the assay to avoid splitting of the UA and xanthine peaks caused by acid deproteinization that could interfere the accurate determination of human plasma XOR activity in our case. Based on the examination of the dependence of XOR activity on added amounts of xanthine and reaction times, the amount of xanthine and reaction time for XOR activity assay were determined to prevent the errors caused by the limiting effect of substrates and plateau phase of the reaction. Using this method, human plasma XOR activities of 25 healthy people were measured. The average human plasma XOR activity was 2.1±0.8 (×10−3 U/ml).
Keywords: Xanthine oxidoreductase; Enzymes;
Sensitive and rapid liquid chromatography–tandem mass spectrometry method for the determination of meloxicam in human plasma by J.L. Wiesner; A.D. de Jager; F.C.W. Sutherland; H.K.L. Hundt; K.J. Swart; A.F. Hundt; J. Els (115-121).
Meloxicam was quantified in human plasma after a single 15 mg oral dose of the drug was given to 26 healthy volunteers. An Applied Biosystems Sciex API 2000 triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, using TurboIonSpray (TIS) in the positive ion mode, was used. Protein precipitation with acetonitrile was followed by C18 reverse phase liquid chromatography and tandem mass spectrometry. The mean recovery for meloxicam was 92% with a lower limit of quantification of 8.96 ng/ml. Piroxicam was used as the internal standard. This assay method makes use of the increased sensitivity and selectivity of tandem mass spectrometry (MS–MS) detection to allow for a more rapid (extraction and chromatography) and selective method for the determination of meloxicam in human plasma than has previously been described.
Determination of valdecoxib and its metabolites in human urine by automated solid-phase extraction–liquid chromatography–tandem mass spectrometry by Ji Y. Zhang; Douglas M. Fast; Alan P. Breau (123-134).
A simple, sensitive and specific automated SPE–LC–MS–MS assay was developed and validated for determination of valdecoxib (I), its hydroxylated metabolite (II) and carboxylic acid metabolite (III) in human urine. The analytes (I, II and III) and a structural analogue internal standard (I.S.) were extracted on a C18 solid-phase extraction cartridge using a Zymark RapidTrace™ automation system. The chromatographic separation was performed on a narrow-bore reverse phase HPLC column with a mobile phase of acetonitrile–water (50:50, v/v) containing 10 mM 4-methylmorpholine (pH 6.0). The analytes were ionized using negative electrospray mass spectrometry, then detected by multiple reaction monitoring with a tandem mass spectrometer. The precursor to product ion transitions of m/z 313→118, m/z 329→196 and m/z 343→196 were used to measure I, II and III, respectively. The assay exhibited a linear dynamic range of 1–200 ng/ml for I and II and 2–200 ng/ml for III in human urine. The lower limit of quantitation was 1 ng/ml for I and II and 2 ng/ml for III. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. Run time of 5.5 min for each sample made it possible to analyze a throughput of 70 human urine samples per run. The assay has been successfully used to analyze human urine samples to support clinical phase I and II studies.
Liquid chromatographic–mass spectrometric determination of cyclooxygenase metabolites of arachidonic acid in cultured cells by Kasem Nithipatikom; Nathan D Laabs; Marilyn A Isbell; William B Campbell (135-145).
A liquid chromatographic–electrospray ionization-mass spectrometric (LC-ESI-MS) technique was developed to simultaneously determine the cyclooxygenase metabolites of arachidonic acid (6-keto-PGF1α, PGD2, PGE2, PGF2α, and PGJ2) produced by cultured cells. Samples were separated on a C18 column with water–acetonitrile mobile phase, ionized by electrospray, and detected in the positive mode. Selected ion monitoring (SIM) of m/z 353, 335, 335, 319, and 317 were used for quantifying 6-keto-PGF1α, PGD2, PGE2, PGF2α, and PGJ2, respectively. Prostaglandins were detected at concentrations as low as 1 pg (S/N=3) on the column. The method was used to determine the production of PGs from bovine coronary artery endothelial cells (ECs) and human prostate cancer cells (PC-3) with different degree of invasiveness. Bradykinin (10−6 M) stimulated a marked increase in the production of 6-keto-PGF1α, PGE2, and PGF2α and a small increase of PGD2 by ECs. 6-Keto-PGF1α was the major metabolite in these cells. The production of PGE2 was threefold higher and PGD2 was twofold higher in PC-3-S (invasive) cells than in PC-3-U (non-invasive) cells.
Keywords: Arachidonic acid; Cyclooxygenase;
Simultaneous determination of free and conjugated bile acids in serum by cyclodextrin-modified micellar electrokinetic chromatography by V.P. Tripodi; S.E. Lucangioli; S.L. Scioscia; C.N. Carducci (147-155).
A simultaneous determination of 15 free and most conjugated forms of bile acids (BA) in serum using capillary electrophoresis is described. The optimized and validated method proposed in this work is straightforward and rapid, employing affordable equipment. A background electrolyte of 5 mM β-cyclodextrin, 5 mM 2-hydroxypropyl-β-cyclodextrin, 50 mM SDS and sodium borate–dihydrogen phosphate pH 7.0 with 10% of acetonitrile was used. The complete separation of 15 BA, not easily achievable with other methods, is performed in less than 12 min using a UV detector with good precision and accuracy. BA were extracted from pretreated serum samples using a C18-solid-phase extraction and the recovery values ranged from 65 to 107.8%. Limits of quantitation were between 0.58 and 3.2 μM. This method proved to be suitable to determine individual BA profiles which are more useful than total serum bile acids as indicators of metabolic disorders and hepatobiliary diseases.
Keywords: Bile acids; Cyclodextrins;
Simultaneous determination of oleuropein and its metabolites in plasma by high-performance liquid chromatography by Anthony Tsarbopoulos; Evangelos Gikas; Nicolaos Papadopoulos; Nektarios Aligiannis; Anthony Kafatos (157-164).
A method based on high-performance liquid chromatography (HPLC) with a diode array detection system was developed and validated aiming at the simultaneous determination of oleuropein (OE) and its metabolites, hydroxytyrosol (HT) and tyrosol (T), in human plasma. These phenolic components are believed to play a vital role in the prevention of coronary artery disease and atherosclerosis. The proposed method includes a clean-up solid-phase extraction procedure (using a C18 column) with high recovery efficiency (85–100%). The statistical evaluation of the method reveals good linearity, accuracy and reproducibility for all the compounds analyzed with RSD values less than 6.5%, while the detection limit is 50 ng/ml for both OE and T and 75 ng/ml for HT. This assay can be employed in bioavailability studies of olive oil phenolic compounds, thus assisting the evaluation of their pharmacological role.
Keywords: Oleuropein; Hydroxytyrosol; Tyrosol;
Determination of thalidomide in transport buffer for Caco-2 cell monolayers by high-performance liquid chromatography with ultraviolet detection by Shufeng Zhou; Yan Li; Phillip Kestell; James W Paxton (165-173).
We report simple validated HPLC methods for the determination of thalidomide in the transport buffer for the human colonic cell line (Caco-2) cell monolayers. An aliquot of 50 μl of the mixture was injected onto a Spherex C18 column (150×4.6 mm; 5 μm) at a flow-rate of 0.5 ml/min of mobile phase consisting of acetonitrile–10 mM ammonium acetate buffer (24:76, v/v, pH 5.5), and thalidomide was detected by ultraviolet detector at a wavelength of 220 nm. Calibration curves for thalidomide were constructed at the concentration range of 0.025–1.0 and 1.0–50 μM in transport buffer. The validated methods were used to determine the transport of thalidomide by Caco-2 monolayers. The transport across the monolayers from the apical (A) to basolateral (B) side was similar to that from B to A side. The apparent permeability coefficient (P app) values of thalidomide at 10–300 μM from the A to B and from B to A side was 2–6×10−5 cm/s, with a marked decrease in P app values from A to B side at increased thalidomide concentration. The A to B transport appears to be dependent on temperature and sodium ion. Sodium azide, 2,4-dinitrophenol (both ATP inhibitors), 5-fluorouracil, cytidine and glutamic acid significantly inhibited the transport of thalidomide. These results indicate that the transport of thalidomide by Caco-2 monolayers was rapid, which might involve an energy-dependent mechanism.
Validation of liquid chromatography assay for the quantitation of (Z)-3-[2,4-dimethyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl]propionic acid (SU006668) in human plasma and its application to a phase I clinical trial by Yasmin Asad; Gillian Cropp; Alison Adams; Anne O’Donnell; Florence Raynaud; Ian Judson; Paul Workman (175-186).
The validation of an analytical method to quantify the antiangiogenic, (Z)-3-[2,4-dimethyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl]propionic acid (SU006668) for pharmacokinetic determination in a phase I clinical trial, is described. HPLC, with a gradient mobile phase and UV detection at 440 nm, was used. SU006668 was extracted from plasma by precipitation of proteins with acetonitrile. The assay was linear from 25 to 2000 ng/ml (r 2=0.997); sensitive (limit of quantification 25 ng/ml), accurate (RE 2.6–11.9%) and reproducible (inter-batch precision C.V. 3.2%). Pharmacokinetic data for six patients are presented. They show linear pharmacokinetics with a low volume of distribution and induction at doses of 50, 100 and 200 mg/m2.
Simultaneous determination of oleuropein and hydroxytyrosol in rat plasma using liquid chromatography with fluorescence detection by Hai-Wei Tan; Kellie L Tuck; Ieva Stupans; Peter J Hayball (187-191).
Oleuropein, the main glycoside present in olives, and hydroxytyrosol, the principal degradation product of oleuropein present in olive oil, have been linked to reduction of coronary heart disease and certain cancers. In the present study a direct and sensitive reversed-phase high-performance liquid chromatographic assay was developed for simultaneous quantification of both oleuropein and hydroxytyrosol. The plasma protein was precipitated with acetonitrile, samples were then centrifuged and supernatants were dried, and reconstituted with water prior to injection. The chromatographic analysis was carried out using a phenyl column and an isocratic elution of acidified water and acetonitrile with fluorescence detection at 281 and 316 nm for excitation and emission, respectively. The calibration curve was linear and limits of quantification were 30 ng/ml and 3 μg/ml for hydroxytyrosol and oleuropein, respectively. The method has been successfully applied to monitor oleuropein and hydroxytyrosol plasma levels in the rat.
Keywords: Oleuropein; Hydroxytyrosol;
Confirmation of ofloxacin precipitation in corneal deposits by microbore liquid chromatography–quadrupole time-of-flight tandem mass spectrometry by Bart A. Sinnaeve; Tineke N. Decaestecker; Ilse J. Claerhout; Philippe Kestelyn; Jean-Paul Remon; Jan F. Van Bocxlaer (193-196).
We investigated the corneal precipitate of a 6-year-old boy with vernal keratoconjunctivitis (VKC), treated with topical ofloxacin 0.3% eyedrops. Because of the extremely small sample amount (corneal scraping), a very sensitive and specific method was needed with the possibility of an unambiguous identification of ofloxacin, supposed to be present in the precipitate. In this respect, tandem Q-TOF mass spectrometry combined with micro LC (1 mm I.D.) was chosen. Confirmation of the presence of ofloxacin in the deposit was obtained by means of the characteristic product ion spectrum produced by CID. This clearly indicated that the precipitate, removed by corneal scraping from the 6-year-old boy with VKC, contained ofloxacin.