Limit Of Quantitation Values Research Articles

Development of an analytical procedure for the determination of emerging and priority organic pollutants in leafy vegetables by pressurized solvent extraction followed by GC-MS determination.

A new multiresidue method for the determination of 13 emerging and priority pollutants in lettuce, including pesticides, pharmaceuticals, personal care products, polycyclic aromatic hydrocarbons (PAHs), and phenolic estrogens, has been developed using matrix solid-phase dispersion combined to pressurized fluid extraction (PFE) followed by gas chromatography coupled to mass spectrometry determination. A sequential optimization strategy based on solvent optimization first, followed by experimental design, was performed in order to maximize target analyte extraction with the aid of response surface methodology. Firstly, a full factorial design was applied to choose the significant variables in PFE; extraction time and temperature were found to have the biggest overall effect on response for most of analytes. They were later optimized performing a central composite design and the variable response of these factors was modeled for all analytes. It was found that marked differences in physicochemical nature exerted a strong influence on extraction conditions and yield. Therefore, the effect of parameters on the response was rather different for some compounds. To overcome this conflicting behavior, a multiple response simultaneous optimization was applied using the desirability function to achieve global optimal operating conditions. The optimal conditions were attained at 13.5 min (two extraction cycles) and 104 degrees C in the PFE by using hexane acetone mixture (1:1). Limit of detection and limit of quantitation values were found to be between 6.6 and 58 and 7.6 and 61.7 microg kg(-1), respectively.

Determination of 7B3 Residues in Cotton Plants by Liquid Chromatography/Mass Spectrometry

A method was developed for the determination of 7B3 (12-propyloxyimino-1,15-pentadecanlactam), a novel macrolactam fungicide, by liquid chromatography/mass spectrometry (LC/MS) with positive electrospray ionization (ESI+). The method used a reversed-phase C18 column and acetonitrile-water (60 + 40, v/v) mobile phase. The quick, easy, cheap, effective, rugged, and safe method was used for extraction of 7B3 from cotton plants, which involved the extraction of 10 g homogenized sample with 10 mL acetonitrile, followed by the addition of 4 g anhydrous MgSO4 and 1.0 g NaCl. After centrifugation, 1 mL of the buffered acetonitrile extract was transferred into a tube containing 50 mg primary secondary amine sorbent and 100 mg anhydrous MgSO4. After shaking and centrifugation, the final extract was transferred to an autosampler vial for concurrent analysis by LC/MS. The results of 7B3 determined by LC/MS in the selective ion monitoring mode were linear, and the matrix effect of the method was evaluated. The average recoveries of 7B3 fortified at different levels were within 84.1-100.2%, and the relative standard deviations were <7.5% for all samples analyzed. The method limit of detection and the limit of quantitation values were 0.03 and 0.1 mg/kg, respectively. The proposed method was successfully applied to determine 7B3 residues in practical samples. This method is sensitive, accurate, reliable, simple, and safe.

Development and validation of a solid-phase extraction and gas chromatography–tandem mass spectrometry method for the determination of isopropyl-9H-thioxanthen-9-one in carton packaged milk

A simple and efficient method for the determination of isopropyl-9H-thioxanten-9-one (ITX) in different fat content milk samples and baby milk samples stored in packaged cartons was developed and validated. Samples were extracted using solid-phase extraction (SPE) and analysed by gas chromatography–tandem mass spectrometry operated in selected reaction monitoring mode (SRM). Validation was carried out in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, precision and trueness. LOD and LOQ values in the low μg/L were achieved, whereas linearity was established within 0.5–500 μg/L range. Good precision was obtained both in terms of intra-day repeatability and inter-day precision on two concentration levels (RSD% lower than 2%). Good percentage recoveries were obtained (92.0–102.0%) even in the presence of high amount of fat. Finally, the developed method was successfully applied to analyse a number of commercial milk samples with different fat content and baby milk samples.

Estimation of Berberine in Ayurvedic Formulations Containing Berberis aristata

A sensitive, simple, rapid, and efficient high-performance thin-layer chromatographic (HPTLC) method has been developed and validated for the analysis of berberine in marketed Ayurvedic formulations containing Berberis aristata DC for regulatory purposes. Chromatography of methanolic extracts of these formulations was performed on silica gel 60 F254 aluminum-backed TLC plates of 0.2 mm layer thickness. The plate was developed up to 66 mm with the ternary-mobile phase butanol-acetic acid-water (8 + 1 + 1, v/v/v) at 33 +/- 5 degrees C with 5 min of tank saturation. The marker, berberine, was quantified at its maximum absorbance of 350 nm. The limit of detection and limit of quantitation values were found to be 5 and 10 ng/spot. The linear regression analysis data for the calibration plot showed a good linear relationship with correlation coefficient = 0.9994 in the concentration range of 10 to 50 ng/spot for berberine with respect to peak area. The instrumental precision was found to be 0.49% coefficient of variation (CV), and repeatability of the method was 0.73% CV. Recovery values from 98.27 to 99.11% indicate excellent accuracy of the method. The developed HPTLC method is very accurate, precise, and cost-effective, and it has been successfully applied to the assay of marketed formulations containing B. aristata for determination of berberine.

Ultraviolet-Photodiode Array and High-Performance Liquid Chromatographic/Mass Spectrometric Studies on Forced Degradation Behavior of Glibenclamide and Development of a Validated Stability-Indicating Method

A forced degradation study on glibenclamide was performed under conditions of hydrolysis, oxidation, dry heat, and photolysis and a high-performance column liquid chromatographic-ultraviolet (HPLC-UV) method was developed to study degradation behavior of the drug under the forced conditions. The degradation products formed under different forced conditions were characterized through isolation and subsequent infrared/nuclear magnetic resonance/mass spectral analyses, or through HPLC/mass spectrometric (HPLC/MS) studies. The drug degraded in 0.1 M HCI and water at 85 degrees C to a major degradation product, 5-chloro-2-methoxy-N-2-(4-sulfamoylphenyl)ethyl]benzamide (III), and to a minor product, 1-cyclohexyl-3-[[4-(2-aminoethyl)-phenyl]sulfonyl]urea (IV). Upon prolonged heating in the acid, the minor product IV disappeared, resulting in formation of 5-chloro-2-methoxy-benzoic acid (II) and an unidentified product (I). Heating of the drug in 0.1 M NaOH at 85 degrees C yielded II and IV as the major products and I and III as the minor products. The drug and the degradation products formed under different conditions were optimally resolved on a C18 column using ammonium acetate buffer (0.025 M, pH 3.5)-acetonitrile (45 + 55) mobile phase at a flow rate of 0.6 mL/min, with detection at 230 nm. The method was validated for linearity, precision, accuracy, and specificity. Limit of detection (LOD) and limit of quantitation (LOQ) values were also determined. The method could be successfully applied for simultaneous quantification of glibenclamide and the major product, III. The response of the method was linear in a narrow [0.4-10 micro/mL, correlation coefficient (r2) = 0.9982] and a wide (0.4-500 microg/mL, r2 = 0.9993) concentration range for glibenclamide, and in the concentration range of 0.025-50 microg/mL (r2 = 0.9998) for III. The method proved to be precise and accurate for both glibenclamide and III. It was specific for the drug and also selective for each degradation product, and LOQ values for the drug were 0.1 and 0.4 microg/mL, whereas those for III were 0.010 and 0.025 microg/mL, respectively.

Design and evaluation of a multi-detection system composed of ultraviolet, evaporative light scattering and inductively coupled plasma mass spectrometry detection for the analysis of pharmaceuticals by liquid chromatography

Reversed-phase liquid chromatography was coupled to a multi-detection system composed of ultraviolet (UV) detection, evaporative laser scattering detection (ELSD) and inductively coupled plasma mass spectrometry (ICP-MS). By applying the principle of post-column solvent compensation, the organic modifier content was kept constant in ELSD and ICP-MS under gradient elution. Chlorine ( 35Cl), bromine ( 79Br and 81Br) and sulfur ( 34S) were monitored in several pharmaceutical compounds. The limit of quantitation (LOQ) was 80 ng/mL for chlorine (chlorpropamide) and 2 ng/mL for bromine (bromazepam). Calibration graphs were linear from 1.0 μg/mL to 100 μg/mL for chlorpropamide ( r 2 0.990) and from 10 ng/mL to 500 ng/mL for bromazepam ( r 2 0.996). The low LOQ value for bromine allows to quantify bromine in pharmaceutical samples below the 0.05% level of the active pharmaceutical ingredient.

Stability-Indicating Thin-Layer Chromatographic Method for Quantitative Determination of Ribavirin

A simple and accurate stability-indicating thin-layer chromatographic (TLC) method is developed and validated for the quantitative determination of ribavirin (RBV) in its bulk and with used for development consists of chloroform-methanol-acetic acid (60:15:15, v/v/v). The separated spots are visualized as bluish green spots after being sprayed with anisaldehyde reagent. RBV is subjected to different accelerated stress conditions. The drug is found to undergo degradation under all stress conditions, and the degradation products are well resolved from the pure drug with significantly different Rf values. The optical densities of the separated spots are found to be linear with the amount of RBV in the range of 5-40 microg/spot with a good correlation coefficient (r=0.9980). The limit of detection and limit of quantitation values are 1.40 and 4.67 microg/spot, respectively. Statistical analysis proves that the method is repeatable and accurate for the determination of RBV in the presence of its degradation products. The method meets the International Conference on Harmonisation/Food and Drug Administration regulatory requirements. The proposed TLC method is successfully applied for the determination of RBV, pure and in capsules, with good accuracy and precision; the label claim percentages are 98.8%+/-1.5%. The results obtained by the proposed TLC method are comparable with those obtained by the official method.

Development and validation of GC–MS method for the determination of methyl methanesulfonate and ethyl methanesulfonate in imatinib mesylate

A gas chromatography–mass spectrometry (GC–MS) method has been developed for the identification and determination of two carcinogenic and genotoxic mesylate esters viz. methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) in imatinib mesylate (INM). The method was optimized based on the peak shapes and resolution of MMS and EMS. The method was validated as per International Conference of Harmonization (ICH) guidelines in terms of limits of detection (LOD), limit of quantitation (LOQ), linearity, precision, accuracy, specificity and robustness. The LOD and LOQ values were found to be 0.3 and 1.0 μg/ml, respectively. The method is linear within the range of 1–15 μg/ml for both the compounds. These mesylate esters were not found in three different batches of pure and pharmaceutical formulations of INM.

Simultaneous determination of salbutamol sulphate, bromhexine hydrochloride and etofylline in pharmaceutical formulations with the use of four rapid derivative spectrophotometric methods

Four simple, rapid, accurate, precise, reliable and economical spectrophotometric methods have been proposed for simultaneous determination of salbutamol sulphate (SS), bromhexine hydrochloride (BH) and etofylline (ET) in pure and commercial formulations without any prior separation or purification. They were first derivative zero crossing spectrophotometry (method 1), simultaneous equation method (method 2), derivative ratio spectra zero crossing method (method 3) and double divisor ratio spectra derivative method (method 4). The ranges for SS, BH and ET were found to be 1–35 μg mL −1, 4–40 μg mL −1 and 5–80 μg mL −1. For methods 1 and 2, the values of limit of detection (LOD) were 0.2314 μg mL −1, 0.4865 μg mL −1 and 0.2766 μg mL −1 and the values of limit of quantitation (LOQ) were 0.7712 μg mL −1, 1.6217 μg mL −1 and 0.9221 μg mL −1 for SS, BH and ET, respectively. For method 3, LOD values were 0.3297 μg mL −1, 0.2784 μg mL −1 and 0.7906 μg mL −1 and LOQ values were 0.9325 μg mL −1, 0.9282 μg mL −1 and 2.6352 μg mL −1 for SS, BH and ET, respectively. For method 4, LOD values were 0.3161 μg mL −1, 0.2495 μg mL −1 and 0.2064 μg mL −1 and LOQ values were 0.9869 μg mL −1, 0.8317 μg mL −1 and 0.6879 μg mL −1 for SS, BH and ET. The precision values were less then 2% R.S.D. for all four methods. The common excipients and additives did not interfere in their determinations. The results obtained by the proposed methods have been statistically compared by means of Student t-test and by the variance ratio F-test.

Porcine follicular fluids: Comparison of solid-phase extraction and matrix solid-phase dispersion for the GC–MS determination of hormones during follicular growth

The capabilities of solid-phase extraction (SPE) and matrix solid-phase dispersion (MSPD) for the determination of the hormones 17β-estradiol, 2-hydroxyestradiol, 4-hydroxyestradiol and 2-methoxyestradiol by gas chromatography–mass spectrometry (GC–MS) in a very complex matrix like porcine follicular fluids were compared, thus proving the highest effectiveness of the SPE technique. Validation was carried out in terms of limit of quantitation (LOQ), precision, accuracy, recovery and stability. LOQ values in the low μg kg −1 were achieved, with all the other parameters satisfying the acceptance criteria for the validation of bioanalytical methods. The applicability of the method to the determination of the hormones in porcine follicular fluids was demonstrated, thus allowing to observe an increase of the concentration of the hormones during the follicular growth.

High performance liquid chromatography/tandem mass spectrometry determination of biogenic amines in typical Piedmont cheeses

The paper presents a new HPLC method, hyphenated with mass spectrometry detection, for the separation and determination of the biogenic amines that are most commonly present in cheese, namely cadaverine, histamine, spermidine, spermine, tyramine and tryptamine. The HPLC–MS/MS method is validated by comparison of the results with those obtained through a literature HPLC–UV determination, based on a pre-column dansyl chloride derivatisation step. The intercalibration is based on the statistical t-test for multiple samples that allows to compare simultaneously the results obtained with the two methods for more analytes and to decide, at a prefixed confidence level, if the two methods are inter-changeable. The new HPLC–MS/MS method, employed in selected reaction monitoring (SRM) mode, permits to achieve for standard solutions limit of detection (LOD) values ranging from 1.7 to 22.5 μg L −1 and LOQ (limit of quantitation) values ranging from 5.6 to 68.2 μg L −1. In order to apply the method in the analysis of cheeses, LOD and LOQ values have also been evaluated in “ricotta” cheese, in order to take as possible into account the matrix interference. In these conditions LODs range between 5.1 and 35.0 μg L −1 and LOQs between 14.2 and 101.2 μg L −1. The whole methodology, comprehensive of the homogenization-extraction process and HPLC–MS/MS analysis, has been applied in the analysis of three typical Piedmont (North-West Italy) cheeses, known as Toma Piemontese, Raschera and Castelmagno.

Rapid HPLC and Direct Flow Injection Analysis Assay for the Determination of Trimetazidine HCl in Pharmaceutical Tablet Formulation

A simple and sensitive high performance liquid chromatographic method (HPLC) and direct flow injection analysis method (FIA) are described for the determination of trimetazidine HCl (TMZ) in tablets. A mobile phase consisting of methanol:0.02 mol · L−1 potassium dihydrogen phosphate:0.005 mol · L−1 sodium dihydrogen phosphate (62:5:33 v/v/v) was used for the resolution of the compound on a reversed phase column for HPLC. For the FIA method; the best solvent system was found to be consisting of methanol:water (10:90 v/v). A flow rate of l.2 mL · min−1 was pumped and active material was detected at 210 nm. Limit of detection (LOD) and limit of quantitation (LOQ) values were found to be 1.2×10−7 mol · L−1 and 3.5×10−7 mol · L−1, respectively, for HPLC. LOD and LOQ values were found to be 2.35×10−7 mol · L−1 and 7.04×10−7 mol · L−1, respectively for FIA. The results obtained from the analysis of TMZ tablet formulations were comparable using both methods.

Determination of Theanine in Commercial Tea by Liquid Chromatography with Fluorescence and Diode Array Ultraviolet Detection

Two liquid chromatographic methods that involve precolumn derivatization with o-phthaladehyde (OPA) and phenylisothiocyanate (PITC) with fluorescence and diode array UV detection for the determination of theanine have been developed. The chromatographic separations were achieved by reverse-phase high-performance liquid chromatography using octadecyl columns and gradient elution. The methods were applied to evaluate the theanine content of commercial tea leaves. The coefficient of variation of the peak area repeatability for within day (n = 8) and between day (n = 8 over 10 days) was lower than 3% for both of the methods. The estimated limit of detection (LOD) and limit of quantitation (LOQ) for the OPA method was 0.12 and 0.35 microg theanine, respectively. The PITC method was 500-fold more sensitive with LOD and LOQ values of 0.25 and 0.75 ng, respectively. The theanine content of the commercial tea samples varied from 2-5 mg/g leaf. The overall % recoveries for these methods ranged from 93-99.3. The sensitivity and simplicity of the method render them suitable for use in quality control laboratories.

Development of a heavy metals enzymatic-based assay using papain

A heavy metals enzymatic-based assay using papain was developed. Papain was assayed using the Casein-coomassie-dye-binding assay. The assay is sensitive to several heavy metals. The IC 50 (concentration of toxicant giving 50% inhibition) of Hg 2+, Ag 2+, Pb 2, Zn 2+ is 0.39, 0.40, 2.16, 2.11 mg l −1, respectively. For Cu 2+ and Cd 2+ the LOQ (limits of quantitation) is 0.004 and 0.1 mg l −1, respectively. The IC 50 and LOQ values were found to be generally comparable to several other enzymatic and bioassays tests such as: immobilized urease, 15-min Microtox™, 48 h Daphnia magna, and 96 h Rainbow trout. The papain assay is xenobiotics tolerant, has a wide pH for optimum activity, is temperature stable, and has a relatively quick assay time. The papain assay was used to identify polluted water samples from industrial sources in Penang, Malaysia. We found one site where the assay gave a positive toxic response. The toxicity of the site was confirmed using Atomic Emission Spectrometry analysis.

Quantification of Gluten Exorphin A5 in cerebrospinal fluid by liquid chromatography–mass spectrometry

In the present work, for the first time, a method for the quantification of the alimentary opioid peptide Gluten Exorphin A5 (GE-A5; Gly-Tyr-Tyr-Pro-Thr) in cerebrospinal fluid (CSF) was developed. Aliquots (5 microL) of CSF were injected into a liquid chromatography-mass spectrometry (LC-MS) instrument equipped with a reversed-phase C18 column at a flow-rate of 0.4 mL/min. The mobile phase consisted of Eluent A water with 0.6% acetic acid as an ion-pairing reagent and Eluent B acetonitrile/methanol (75:25, v/v). The LC-MS system was programmed to divert column flow to waste for 4 min after injection, after which time flow was directed into the mass spectrometer that operated in positive ion mode. No significant interfering peaks were detected at the retention times of GE-A5 in CSF blanks. The lower limit of detection and the lower limit of quantitation values for GE-A5 in CSF were established at 0.60 and 1.50 ng/mL, respectively. The intra- and inter-day precision values were <5% relative standard deviation. The intra- and inter-day accuracy were 99.6-102.8% and 100.0-101.9%, respectively. The reported assay employs extremely small volumes of CSF, thus allowing the analysis of GE-A5 from both small and large animal models.

Development and validation of a solid phase micro-extraction–gas chromatography–mass spectrometry method for the determination of furan in baby-food

An efficient and simple method for the determination of furan in baby-food (vegetables and fruits) by solid phase micro-extraction–gas chromatography–mass spectrometry (SPME–GC–MS) was developed and validated. Experimental design was used to investigate the effects of temperature and time of extraction. The calculated regression model was used to find the experimental conditions providing the optimal SPME extraction yield. Validation was carried out in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, precision and trueness. LOD and LOQ values in the low ng kg −1 were achieved, whereas linearity was established over two order of magnitude. Good precision was obtained both in terms of intra-day repeatability and between-day precision on two concentration levels (RSD% lower than 3.6%). Recovery values of 91.5 ± 6.2% and of 96.1 ± 1.3% ( n = 3) were calculated at 75 ng kg −1 and 75 μg kg −1 level. Finally, the applicability of the method to the determination of furan in a number of commercial and home-made baby-food samples was demonstrated.

Bioremediation of Toluene and Naphthalene: development and Validation of a GC‐FID Method for Their Monitoring

Bioremediation of toluene and naphthalene in liquid cultures of bacteria grown in the presence of these aromatic compounds as unique sources of carbon was investigated by gas chromatography (GC). For this purpose, a method based on the use of GC with flame ionization detection was developed and validated. Validation was carried out in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, precision and trueness. In the case of naphthalene, LOD and LOQ values of 0.43 and 0.72 mg kg(-1) were achieved. Linearity was established over one order of magnitude in the range of interest, i.e. 10-100 mg kg(-1). Excellent precision was obtained both in terms of intra-day repeatability and between-day precision on two concentration levels (RSD% lower than 0.5%). A recovery of 97.9 +/- 0.2% (n=3) was calculated by addition of 640 mg kg(-1) of naphthalene to the Bushnell & Haas mineral salts basal solution containing the micro-organisms. Findings clearly showed a reduction of the naphthalene content equal to 50% and 75% after two and four weeks of contact with the micro-organisms, whereas a lower degradation was shown in the case of toluene. Finally bioremediation activity was ascribed to two different microbial populations, Bordetella Petrii and Bacillus Sphericus, which survived in the polluted medium.

ACETATE AND CHLORIDE DETERMINATION BY ION EXCHANGE HPLC WITH INDIRECT PHOTOMETRIC DETECTION AND ITS APPLICATION TO A β-METHYLCARBAPENEM ANTIBIOTIC

A high performance liquid chromatographic (HPLC) method, using an anion exchange column with indirect photometric detection, was developed for the determination of acetate and chloride anions in the antibiotic ertapenem sodium. HPLC with indirect photometric detection was used due to the UV-transparent properties of acetate and chloride and the advantage that the method employs simple rugged laboratory chromatographic equipment. The challenges encountered with method optimization are discussed. The method gives baseline separation of the anions with limit of quantitation values of 0.005 wt % and 0.006 wt % for acetate and chloride, respectively. The method was also validated for precision, linearity, recovery, ruggedness, and solution stability.

Comparative analytical quantitation of clenbuterol in biological matrices using GC-MS and EIA.

A simple and sensitive procedure utilizing GC-MS for the identification and quantitation of clenbuterol in biofluids and tissues is described. This improved method utilizes trimethylboroxine for the derivatization of clenbuterol, requires only 1 mL/g of biological sample, and most importantly does not require an extra cleaning step for urine specimens prior to extraction. Linear quantitative response curves have been generated for derivatized clenbuterol over a concentration range of 5-200 ng/mL. The extraction efficiency at four representative points of the standard curve exceeded 90% in both specimen types (plasma and urine). Linear regression analyses of the standard curve in both specimen types exhibited correlation coefficients ranging from 0.997 to 1.000. The Limit of detection (LOD) and Limit of quantitation (LOQ) values for plasma specimens were determined to be 0.5 and 1.5 ng/mL respectively. For urine specimens, LOD and LOQ values were 0.2 and 0.7 ng/microL respectively. Percentage recoveries ranged from 91 to 95% for urine and 89 to 101% for plasma. Precision and accuracy (within-run and between-run) studies reflected a high level of reliability and reproducibility of the method. In addition to its reliability, sensitivity and simplicity, this modified procedure is more efficient and cost effective, requiring less time, only 1 mL of sample, and minimal amounts of extraction solvents. The applicability of the method for the detection and quantitation of clenbuterol in biological tissues of rats treated with the drug was demonstrated successfully. For comparative analysis of clenbuterol in plasma and liver samples, both GC-MS and enzyme immunoassay (EIA) methods are found to be suitable. Due to potential antibody-cross reactivity with EIA, the GC-MS method is the method of choice for most samples because of its specificity. However, the EIA method is considered the method of choice for analysis of clenbuterol found in concentrations below the limits of quantitation by GC-MS due to its sensitivity.

Simultaneous determination of four anthracyclines and three metabolites in human serum by liquid chromatography–electrospray mass spectrometry

A sensitive and very specific method, using liquid chromatography–electrospray mass spectrometry (LC–ES-MS), was developed for the determination of epirubicin, doxorubicin, daunorubicin, idarubicin and the respective active metabolites of the last three, namely doxorubicinol, daunorubicinol and idarubicinol in human serum, using aclarubicin as internal standard. Once thawed, 0.5-ml serum samples underwent an automated solid-phase extraction, using C 18 Bond Elut cartridges (Varian) and a Zymark Rapid-Trace robot. After elution of the compounds with chloroform–2-propanol (4:1, v/v) and evaporation, the residue was reconstituted with a mixture of 5 m M ammonium formate buffer (pH 4.5)–acetonitrile (60:40, v/v). The chromatographic separation was performed using a Symmetry C 18, 3.5 μm (150×1 mm I.D.) reversed-phase column, and a mixture of 5 m M ammonium formate buffer (pH 3)–acetonitrile (70:30, v/v) as mobile phase, delivered at 50 μl/min. The compounds were detected in the selected ion monitoring mode using, as quantitation ions, m/ z 291 for idarubicin and idarubicinol, m/ z 321 for daunorubicin and daunorubicinol, m/ z 361 for epirubicin and doxorubicin, m/ z 363 for doxorubicinol and m/ z 812 for aclarubicin (I.S.). Extraction recovery was between 71 and 105% depending on compounds and concentration. The limit of detection was 0.5 ng/ml for daunorubicin and idarubicinol, 1 ng/ml for doxorubicin, epirubicin and idarubicin, 2 ng/ml for daunorubicinol and 2.5 ng/ml for doxorubicinol. The limit of quantitation (LOQ) was 2.5 ng/ml for doxorubicin, epirubicin and daunorubicinol, and 5 ng/ml for daunorubicin, idarubicin, doxorubicinol and idarubicinol. Linearity was verified from these LOQs up to 2000 ng/ml for the parent drugs ( r≥0.992) and 200 ng/ml for the active metabolites ( r≥0.985). Above LOQ, the within-day and between-day precision relative standard deviation values were all less than 15%. This assay was applied successfully to the analysis of human serum samples collected in patients administered doxorubicin or daunorubicin intravenously. This method is rapid, reliable, allows an easy sample preparation owing to the automated extraction and a high selectivity owing to MS detection.