Headspace solid-phase microextraction in combination with gas chromatography and tandem mass spectrometry for the determination of organochlorine and organophosphorus pesticides in whole numan blood

A method for the determination of several organochlorine and organophosphorus pesticides in human whole blood samples was developed. The combination of solid-phase microextraction in headspace mode with gas chromatography with tandem mass spectrometry allowed the determination of 11 selected pesticides at ppb levels, minimizing the sample treatment. Quantitation was carried out by means of calibration curves prepared in blood using labelled surrogate/internal standards. The method showed good linearity between 1 and 50 ng ml(-1) (0.5-25 ng ml(-1) for HCB) using second-order calibration curves. Precision was found to be better than 20% at the three concentration levels assayed in the range of ng ml(-1). The detection limits obtained were in the range 0.02-0.7 ng ml(-1), except for p,p'-DDT (3 ng ml(-1)). The developed procedure was applied to blood and serum samples obtained from agricultural workers. HCB. beta-HCH and p,p'-DDE were most frequently detected in the samples analyzed.     

Validation of a liquid chromatographic-tandem mass spectrometric method for the measurement of (R)-1-[2,3-dihydro-2-oxo-1-pivaloylmethyl-5-(2-pyridyl)-1H-1,4-benzodiazepin-3-yl]-3-(3-methylaminophenyl)urea (YF476) in human plasma

A sensitive and specific liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method has been validated for the measurement of YF476 in human plasma. The method involves a simple liquid-liquid extraction procedure, chromatography of the extracts on a C(18) column, atmospheric pressure chemical ionisation and detection in the multiple reaction monitoring mode. The calibration line was linear over the concentration range 0.1 ng/ml (the limit of quantification) to 25.0 ng/ml. Intra- and inter-batch precision was <14% and intra- and inter-batch accuracy was <11% over the entire calibration range. The bioanalytical method is robust and has been used for the analysis of many samples from human subjects involved in early clinical studies (Phase I).     

Determination of Tirofiban in human serum by liquid chromatography-tandem mass spectrometry

A liquid chromatography-tandem mass spectrometric (LC-MS-MS) method with a rapid and simple sample preparation was developed and validated for the determination of Tirofiban in biological fluids. Tirofiban in serum samples was extracted and cleaned up by using an automated solid phase extraction method. An external calibration was used. The mass spectrometer was operated in the multiple reaction monitoring mode (MRM). A good linear response over the range of 2-200ng/ml was demonstrated. The accuracy for Tirofiban ranged from 94.8 to 110.8% within-day and from 103.0 to 104.7% between-day. The lower limit of quantification was 2ng/ml. This method is suitable for pharmacokinetic studies.     

A liquid chromatographic-tandem mass spectrometric method for determination of artesunate and its metabolite dihydroartemisinin in human plasma

A bioanalytical method for the analysis of artesunate and its metabolite dihydroartemisinin in human plasma using high throughput solid-phase extraction in the 96-wellplate format and liquid chromatography coupled to positive tandem mass spectroscopy has been developed and validated. The method was validated according to published FDA guidelines and showed excellent performance. The within-day and between-day precisions expressed as RSD, were lower than 7% at all tested concentrations including the lower limit of quantification. Using 50 microl plasma the calibration range was 1.19-728 ng/ml with a limit of detection at 0.5 ng/ml for artesunate and 1.96-2500 ng/ml with a limit of detection at 0.6 ng/ml for dihydroartemisinin. Using 250 microl of plasma sample the lower limit of quantification was decreased to 0.119 ng/ml for artesunate and 0.196 ng/ml dihydroartemisinin. Validation of over-curve samples in plasma ensured that accurate estimation would be possible with dilution if samples went outside the calibration range. The method was free from matrix effects as demonstrated both graphically and quantitatively.     

Headspace solid-phase microextraction and capillary gas chromatographic-mass spectrometric determination of rivastigmine in canine plasma samples

A simple, rapid and sensitive method for determination of rivastigmine in plasma samples was developed using headspace solid-phase microextraction (HS-SPME) and gas chromatography with mass spectrometry (GC-MS). The optimum conditions for the SPME procedure were: headspace extraction on a 65-microm polydimethylsiloxane/divinylbenzene (PDMS/DVB) fiber; 0.5 ml of plasma modified with 1.0 ml of sodium hydroxide-sodium carbonate solution (0.7 M:0.5M); extraction temperature of 100 degrees C, with stirring at 2000 rpm for 30 min. The calibration curve showed linearity in the range from 0.2 to 80 ng/ml with regression coefficient corresponding to 0.9965 and coefficient of the variation of the points of the calibration curve lower than 10%. The quantification limit for rivastigmine in plasma was 0.2 ng/ml. The method was applied to determination of rivastigmine in canine plasma samples from animals after a single oral administration.     

Selective determination of sultopride in human plasma using high-performance liquid chromatography with ultraviolet detection and particle beam mass spectrometry

We developed a sensitive and selective method for determining levels of sultopride, a neuroleptic drug of the substituted benzamide, in human plasma using high-performance liquid chromatography (HPLC) combined with UV detection and particle beam mass spectrometry (PBMS). Sutopride was extracted with tert.-butylmethyl ether using a salting-out technique. Tiapride served as an internal standard (I.S.). Sutopride and I.S. were separated by HPLC on a silica column with a mobile phase of acetonitrile-0.1 M ammonium acetate (94:6, v/v). The calibration curves were linear over the concentration range from 5 to 1000 ng/ml by HPLC with UV detection and from 10 to 1000 ng/ml with PBMS detection. The limit of quantitation was 5 ng/ml with UV detection and 10 ng/ml with PBMS detection. The absolute recovery was 92% and the within-day coefficients of variation were 2.9–7.1% at plasma concentrations from 50 to 500 ng/ml, determined by HPLC with UV detection. Using this method, we measured the plasma concentrations of sultopride with replicate analyses in four hospitalized patients and steady-state plasma levels were determined to be 161.6±30.8, 321.1±93.7, 726.5±143.1 and 1273.6±211.2 ng/ml, respectively.     

A highly sensitive LC-MS-MS assay for analysis of midazolam and its major metabolite in human plasma: applications to drug metabolism

Betamethasone is a synthetic corticosteroid designed to exert a marked glucocorticoid activity. As the free alcohol, betamethasone finds widespread clinical applications related to its anti-inflammatory and immunosuppressant activity. In the present study, a fast, sensitive, robust method was developed for the determination and quantification of betamethasone in human plasma by liquid chromatography coupled with tandem mass spectrometry, using photospray ionization in negative mode. Betamethasone was extracted from 0.5 ml human plasma by liquid-liquid extraction (LLE) using chloramphenicol as internal standard. The method has a chromatographic run of 2.5 min using a C(18) analytical column (100 mm x 2.1 mm i.d.) and the linear calibration curve over the range was linear from 0.05 to 50 ng ml(-1) (r(2)>0.993). The between-run precision, based on the relative standard deviation replicate quality controls was 94.1% (0.15 ng ml(-1)), 90.7% (4.0 ng ml(-1)) and 97.2% (40 ng ml(-1)). The between-run accuracy for the above-mentioned concentrations was 11.9, 9.0 and 9.8%, respectively. The method herein described was employed in a bioequivalence study of two formulations of dexchlorpheniramine/betamethasone 2 mg/0.25 mg tablets.     

Sensitive determination of pethidine in body fluids by gas chromatography-tandem mass spectrometry

We have presented a simple and sensitive method for determining pethidine, a narcotic analgesic drug in body fluids by gas chromatography-tandem mass spectrometry (GC-MS/MS). Pethidine and 4'-piperidinoacetophenone (internal standard) were extracted from body fluids with Bond Elut C(18) columns; the recoveries were above 85% for both compounds. The calibration curves for blood and urine showed good linearities in the range of 1.25-40 ng/ml. Its detection limits (signal-to-noise ratio=3) were estimated to be approximately 0.5 ng/ml of whole blood and urine.     

Quantitative trace analysis of estriol in human plasma by negative ion chemical ionization gas chromatography-mass spectrometry using a deuterated internal standard

A stable isotope dilution gas chromatography-mass spectrometry (GC-MS) assay for the trace level determination of estriol in human plasma is described. Negative ion chemical ionization (NICI) MS is used for highly specific detection. The method involves derivatization of the phenolic hydroxyl to the pentafluorobenzyl ether derivative and subsequent reaction of the remaining hydroxyls with heptafluorobutyric anhydride. This derivative allows detection of the strikingly abundant phenolate ion under NICI conditions. [2,4,17beta]-2H(3)-labeled estriol was used as an internal standard. For high-level measurements (>313 ng/l) plasma was directly derivatized by extractive alkylation followed by heptafluorobutylation prior to analysis. A rapid and simple sample work up procedure was elaborated for trace level determinations (>5 ng/l plasma) using solid-phase extraction on C(18) with an absolute recovery of 92.9%. For low-level measurements, the calibration curve was linear in the range of 5 to 625 ng/l (r=0.99993). Inter-assay analytical precisions (RSDs) were 1.29, 2.30 and 2.89% at 39, 156 and 650 ng/l plasma, respectively. For high-level measurements, calibration curve linearity was observed in the range of 0.313 to 20 microg/l (r=0.99998). Inter-assay analytical precisions (RSDs) were 5.17, 1.92, 2.57 and 2.74% at 0.313, 0.625, 2.5 and 10 microg/l plasma, respectively. Postmenopausal plasma was used for spiked plasma samples. Sensitivity and specificity of the presented method allows adequate determination of estriol in human plasma samples.     

Quantification of the bombesin/gastrin releasing peptide antagonist RC-3095 by liquid chromatography-tandem mass spectrometry

Bombesin (BN) and its mammalian equivalent, gastrin-releasing peptide (GRP), stimulate cell proliferation and are involved in the pathogenesis of several types of human cancer. BN/GRP and their receptors were shown to be critical for the growth of various human malignancies, such as small-cell lung, prostate, ovary, stomach and breast cancers in the human tumor xenograft model. In the present study, a fast, sensitive, robust method was developed for the determination and quantification of a BN/GRP receptor antagonist RC-3095 (D-Tpi-Gln-Trp-Ala-Val-Gly-His-Leupsi(CH2NH)Leu-NH2), in human plasma by liquid chromatography coupled with tandem mass spectrometry. RC-3095 was extracted from 0.2 ml human plasma by protein precipitation using cold acetonitrile (0.4 ml). The method has a chromatographic run of 10 min using a C(8) analytical column (150 mm x 4.6 mm i.d.) and the linear calibration curve over the range was linear from 20 to 10000 ng ml(-1) (r(2)>0.994). The between-run precision, based on the relative standard deviation replicate quality controls, was 5.7% (60 ng ml(-1)), 7.1% (600 ng ml(-1)) and 6.8% (8000 ng ml(-1)). The between-run accuracy was +/-0.0, 2.1 and 3.1% for the above-mentioned concentrations, respectively. The developed procedure allows the quantitative determination of peptide RC-3095 for pharmacokinetics studies in human plasma.     

Liquid chromatography-tandem mass spectrometry assay for human serum testosterone and trideuterated testosterone

A liquid chromatography tandem mass spectrometry assay for serum testosterone (T) and trideuterated testosterone (d(3)T) was developed in order to support clinical research studies that determine the pharmacokinetics, production rate, and clearance of testosterone by administration of trideuterated testosterone. After adding 19-nortestosterone as the internal standard (I.S.), sodium acetate buffer, and ether, to a serum aliquot, the mixture was shaken and centrifuged, and the ether was dried. The extract was reconstituted in methanol and 15 microl was injected into a liquid chromatograph equipped with an autosampler and Applied Biosystems-Sciex API 300 triple quadrupole mass spectrometer operated in the positive ion mode. T, d(3)T, and I.S. were monitored with transitions m/z 289 to m/z 97, m/z 292 to m/z 97, and m/z 275 to m/z 109, respectively. The two calibration curves were linear over the entire measurement range of 0-20 ng/ml for T and 0-2.0 ng/ml for d(3)T. The LOQs for T and d(3)T were 0.5 ng/ml and 0.05 ng/ml. The recoveries for T and d(3)T were 91.5 and 96.4%. For T at 1.25 ng/ml and 4.0 ng/ml, the intra-day precision (RSD, %) was 3.9 and 4.3% and intra-day accuracy 0.01 and 4.5%, respectively. The inter-day precision at these levels was 5.3 and 5.4% and inter-day accuracy was 1.9 and 0.3%. For d(3)T at 0.125 ng/ml and 0.4 ng/ml, the intra-day precision (RSD, %) was 2.8 and 8.3% and intra-day accuracy was 1.8 and 5.6%. The inter-day precision at these levels was 10.0 and 7.6% and inter-day accuracy was 5.7 and 3.4%. The concentrations of T in the 38 healthy subjects ranged from 2.5 to 14.0 ng/ml (mean 6.2 ng/ml).     

Improved solid-phase extraction and HPLC measurement of torasemide and its important metabolites

Torasemide is a "loop type" diuretic drug. For pharmacokinetic studies sensitive analytic methods are essential for authentic qualitative and quantitative information. A robust, selective and sensitive HPLC method is described for the simultaneous determination of torasemide, its major metabolite M5 and its active metabolites M1 and M3 and an internal standard within 17 min. Solid-phase extraction with C(2)-cartridges was used for the clean-up of plasma samples. The chromatographic separation was carried out on a CN-column with a mobile phase consisting of perchloric acid (0.02 M, pH 2.5)/acetonitrile (90/10, v/v)). The calibration range used reached from 20 to 1000 ng/ml for all analytes. Coefficients of variation were less than 10% at every calibration point for each analyte. Plasma concentrations in samples obtained from volunteers in the course of a clinical study could be reliably measured with this method. Median maximum concentrations in plasma after a 10mg oral dose during a 24h study interval were located at 1h for torasemide, 1h for M1 and 2h for M5. Concentrations between 2226 and <20 ng/ml for torasemide, between 159 and <20 ng/ml for M1 and between 420 and <20 ng/ml for M5 were observed.     

Determination of nimodipine in human plasma by a sensitive and selective liquid chromatography-tandem mass spectrometry method

A sensitive and highly selective liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed to determine nimodipine in human plasma. The analyte and internal standard nitrendipine were extracted from plasma samples by n-hexane-dichloromethane-isopropanol (300:150:4, v/v/v), and chromatographed on a C(18) column. The mobile phase consisted of methanol-water-formic acid (80:20:1, v/v/v). Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via atmospheric pressure chemical ionization (APCI) source. The method has a limit of quantification of 0.24 ng/ml. The linear calibration curves were obtained in the concentration range of 0.24-80 ng/ml. The intra- and inter-day precisions were lower than 4.4% in terms of relative standard deviation (R.S.D.), and the accuracy ranged from 0.0 to 5.8% in terms of relative error (RE). This validated method was successfully applied for the evaluation of pharmacokinetic profiles of nimodipine tablets administered to 18 healthy volunteers.     

Development and validation of a rapid HPLC method for simultaneous determination of tramadol, and its two main metabolites in human plasma

Tramadol, an analgesic agent, and its two main metabolites O-desmethyltramadol (M1) and N-desmethyltramadol (M2) were determined simultaneously in human plasma by a rapid and specific HPLC method. The sample preparation was a simple extraction with ethyl acetate. Chromatographic separation was achieved with a Chromolith Performance RP-18e 50 mm x 4.6 mm column, using a mixture of methanol:water (13:87, v/v) adjusted to pH 2.5 by phosphoric acid, in an isocratic mode at flow rate of 2 ml/min. Fluorescence detection (lambda(ex)=200 nm/lambda(em)=301 nm) was used. The calibration curves were linear (r(2)>0.997) in the concentration range of 2.5-500 ng/ml, 1.25-500 ng/ml and 5-500 ng/ml for tramadol, M1 and M2, respectively. The lower limit of quantification was 2.5 ng/ml for tramadol, 1.25 ng/ml for M1 and 5 ng/ml for M2. The within- and between-day precisions in the measurement of QC samples at four tested concentrations were in the range of 2.5-9.7%, 2.5-9.9% and 5.9-11.3% for tramadol, M1 and M2, respectively. The developed procedure was applied to assess the pharmacokinetics of tramadol and its two main metabolites following administration of 100mg single oral dose of tramadol to healthy volunteers.     

Development and validation of a liquid chromatography-tandem mass spectrometric assay for pitavastatin and its lactone in human plasma and urine

A rapid, selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with electrospray ionization (ESI) was developed and validated for the simultaneous determination of pitavastatin and its lactone in human plasma and urine. Following a liquid-liquid extraction, both the analytes and internal standard racemic i-prolact were separated on a BDS Hypersil C(8) column, using methanol-0.2% acetic acid in water (70: 30, v/v) as the mobile phase. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode using the transition m/z 422.4-->m/z 290.3 for pitavastatin, m/z 404.3-->m/z 290.3 for pitavastatin lactone and m/z 406.3-->m/z 318.3 for the internal standard, respectively. Linear calibration curves of pitavastatin and its lactone were obtained in the concentration range of 1-200 ng/ml, with a lower limit of quantitation of 1 ng/ml. The intra- and inter-day precision values were less than 4.2%, and accuracies were between -8.1 and 3.5% for both analytes. The proposed method was utilized to support clinical pharmacokinetic studies of pitavastatin in healthy subjects following oral administration.     

Simultaneous determination of flupirtine and its major active metabolite in human plasma by liquid chromatography–tandem mass spectrometry

A rapid, selective and sensitive HPLC–tandem mass spectrometry method was developed and validated for simultaneous determination of flupirtine and its active metabolite D-13223 in human plasma. The analytes and internal standard diphenhydramine were extracted from plasma samples by liquid–liquid extraction, and chromatographed on a C₁₈ column. The mobile phase consisted of acetonitrile–water–formic acid (60:40:1, v/v/v), at a flow rate of 0.5 ml/min. Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via atmospheric pressure chemical ionization (APCI). The method has a limit of quantitation of 10 ng/ml for flupirtine and 2 ng/ml for D-13223, using 0.5-ml plasma sample. The linear calibration curves were obtained in the concentration range of 10.0–1500.0 ng/ml for flupirtine and 2.0–300.0 ng/ml for D-13223. The intra- and inter-run precision (RSD), calculated from quality control (QC) samples was less than 7.2% for flupirtine and D-13223. The accuracy as determined from QC samples was less than 5% for the analytes. The overall extraction recoveries of flupirtine and D-13223 were determined to be about 66% and 78% on average, respectively. The method was applied for the evaluation of the pharmacokinetics of flupirtine and active metabolite D-13223 in volunteers following peroral administration.     

Determination of the enantiomers of ketamine and norketamine in human plasma by enantioselective liquid chromatography-mass spectrometry

A sensitive enantioselective liquid chromatographic assay with mass spectrometric detection has been developed and validated for the simultaneous determination of plasma concentrations of (R)- and (S)-ketamine, and (R)- and (S)-norketamine. The compounds were extracted from human plasma using solid-phase extraction and then directly injected into the LC-MS system for detection and quantification. Enantioselective separations were achieved on a liquid chromatographic chiral stationary phase based upon immobilized alpha(1)-acid glycoprotein (the Chiral AGP column). The separations were achieved using a mobile phase composed of 2-propanol-ammonium acetate buffer (10 mM, pH 7.6) (6:94, v/v), a flow-rate of 0.5 ml/min and a temperature of 25 degrees C. Under these conditions, the analysis time was 20 min. Detection of the ketamine, norketamine and bromoketamine (internal standard) enantiomers was achieved using selected ion monitoring at m/z 238.1, 224.1 and 284.0, respectively. Extracted calibration curves were linear from 1 to 125 ng/ml per enantiomer for each analyte with correlation coefficients better than 0.9993 and intra- and inter-day RSDs of less than 8.0%. The method was applied to samples from a clinical study of ketamine in pain management.     

Simultaneous determination of steroid composition of human testicular fluid using liquid chromatography tandem mass spectrometry

A rapid, sensitive, and specific method using liquid chromatography tandem mass spectrometry (LC/MS/MS) has been developed for simultaneous determination of testosterone (T), dihydrotestosterone (DHT), estradiol (E2), and 5alpha-androstan-3alpha, -17beta-diol (3alpha-Diol) within human testicular fluid. Sample pretreatment involved a one-step extraction with diethyl ether. The analytes were separated on a Waters X-Terra C18 (150 mm x 2.1 mm i.d., 3.5 microm) analytical column with acetonitrile/water mobile phase (70:30, v/v) containing 0.1% formic acid using isocratic flow at 0.15 ml/min for 8 min. The column effluent was monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 0.1-50 ng/ml for T, 0.02-1 ng/ml for DHT, 0.05-2 ng/ml for E2, and 0.2-10 ng/ml for 3alpha-Diol, with values for the coefficient of determination of >0.99. The overall extraction efficiency was greater than 86% for T, 75% for DHT, 66% for E2, and 60% for 3alpha-Diol. The values for within-day and between-day precision and accuracy were <15%. We measured each of the four steroids in testicular sample volumes of only 20 microl, obtained by percutaneous testicular aspiration. The mean intratesticular testosterone concentration found by LC/MS/MS, 572 +/- 102 ng/ml, was similar to that previously obtained by radioimmunoassay (RIA). The mean intratesticular estradiol concentration was 15.7 +/- 2.3 ng/ml, which also correlated well with RIA measurement. Both DHT and 3alpha-Diol were below the limits of detection by RIA, but could be measured accurately by LC/MS/MS. In conclusion, LC/MS/MS represents a sensitive and accurate means by which to measure four separate steroids within small volume samples of testicular fluid.     

Simultaneous determination of glipizide and rosiglitazone unbound drug concentrations in plasma by equilibrium dialysis and liquid chromatography-tandem mass spectrometry

Glipizide and rosiglitazone are widely used to treat Type 2 diabetes. In order to investigate drug-drug protein binding interaction between glipizide and rosiglitazone, a method was developed and validated for simultaneously determining the free (unbound) fraction of glipizide and rosiglitazone in plasma employing equilibrium dialysis for the separation of free drug and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for quantitation. Post-dialysis human plasma or buffer samples of 0.2 ml were extracted using a liquid-liquid extraction procedure and analyzed by a high performance liquid chromatography electrospray tandem mass spectrometer system. The compounds were eluted isocratically on a Zorbax SB-Phenyl column, ionized using an atmospheric pressure electrospray ionization source and analyzed in positive ion mode with multiple reaction monitoring. The ion transitions monitored were m/z 446-->321 for glipizide, m/z 358-->135 for rosiglitazone, and m/z 271-->155 for tolbutamide (internal standard, IS). The chromatographic run time was 5 min per injection, with retention times of 2.3, 3.4 and 2.3 min for glipizide, rosiglitazone and IS, respectively. The calibration curves of glipizide and rosiglitazone were over the range of 1-2000 ng/ml (r(2)>0.9969) in the combined matrix of human plasma and isotonic sodium phosphate buffer (1:1, v/v). The inter-assay precision and accuracy of the quality control samples were <10.9% of coefficient of variability and >93.5% and 94.5% of nominal concentration for glipizide and rosiglitazone, respectively. The lower limit of quantitation of both glipizide and rosiglitazone was 1.0 ng/ml. Both glipizide and rosiglitazone bound to plasma protein extensively (>99% bound). Glipizide and rosiglitazone free fraction averaged 0.678+/-0.071 and 0.389+/-0.061%, respectively, at plasma concentration of 1000 ng/ml. This developed method proves reproducible and sensitive and its application to clinical samples is also reported.     

Simple high-performance liquid chromatographic method for the determination of ranitidine in human plasma

A simple high-performance liquid chromatographic method was developed for the determination of ranitidine in human plasma. Prior to analysis, ranitidine and the internal standard (metoprolol) were extracted from alkalinized plasma samples using dichloromethane. The mobile phase was 0.05 M potassium dihydrogenphosphate–acetonitrile (88:12, v/v) adjusted to pH 6.5. Analysis was run at a flow-rate of 1.3 ml/min and at a detection wavelength of 229 nm. The method is sensitive with a detection limit of 1 ng/ml at a signal-to-noise ratio of 3:1, while the quantification limit was set at 15 ng/ml. The calibration curve was linear over a concentration range of 15–2000 ng/ml. Mean recovery value of the extraction procedure was about 90%, while the within-day and between-day coefficients of variation and percent error values of the assay method were all less than 15%.