Identification and quantification of 8 sulfonylureas with clinical toxicology interest by liquid chromatography-ion-trap tandem mass spectrometry and library searching.Sulfonylureas are a wide group of compounds used for different purposes. Some have been used for more than 5 decades as antidiabetic drugs for the treatment of hyperglycemia hyperglycemia: see diabetes. in patients with diabetes mellitus diabetes mellitus
Disorder of insufficient production of or reduced sensitivity to insulin. Insulin, synthesized in the islets of Langerhans (see Langerhans, islets of), is necessary to metabolize glucose. In diabetes, blood sugar levels increase (hyperglycemia). type II. The misuse of Sulfonylureas, however, can lead to hypoglycemia hypoglycemia: see diabetes.
Below-normal levels of blood glucose, quickly reversed by administration of oral or intravenous glucose. Even brief episodes can produce severe brain dysfunction. (1), including unexplained severe hypoglycemia in some patients with Munchausen syndrome. To aid in the differentiation of drug misuse vs other etiologies, such as insulinoma, evaluation of repetitive hypoglycemic hypoglycemic /hy·po·gly·ce·mic/ (-gli-sem´ik)
1. pertaining to, characterized by, or causing hypoglycemia.
2. an agent that lowers blood glucose levels. crises of unknown origin should include testing to assess whether the patient has taken a sulfonylurea sulfonylurea /sul·fo·nyl·urea/ (sul?fo-nil-u-re´ah) any of a class of compounds that exert hypoglycemic activity by stimulating the islet tissue to secrete insulin; used to control hyperglycemia in patients with type 2 diabetes mellitus drug. Failure to identify drug-induced hypoglycemia may lead to exploratory surgery or even subtotal subtotal /sub·to·tal/ (sub-to´t'l) less than, but often almost, complete. pancreatectomy Pancreatectomy Definition
Pancreatectomy is the surgical removal of the pancreas. Pancreatectomy may be total, in which case the whole organ is removed, or partial, referring to the removal of part of the pancreas. (2).
Several analytical methods for the screening and measurement of Sulfonylureas in biological fluids have been described. Most methods are based on HPLC HPLC high-performance liquid chromatography.
high performance liquid chromatography.
HPLC High-performance liquid chromatography Lab instrumentation A highly sensitive analytic method in which analytes are placed with ultraviolet (3), diode array (4), or fluorescence detection after derivatization of serum extracts (5). A micellar electrokinetic capillary chromatographic chro·mat·o·graph
An instrument that produces a chromatogram.
tr.v. chro·mat·o·graphed, chro·mat·o·graph·ing, chro·mat·o·graphs
To separate and analyze by chromatography. method with ultraviolet detection has also been proposed for the detection of Sulfonylureas in urine (6). These methods may lack specificity, particularly when a single ultraviolet wavelength is used, which may cause false positives. Paroni et al. (7) published an interesting capillary electrophoresis method but clearly mentioned that it should not be used alone to give a definitive diagnosis of intake of these drugs. More recently, higher specificity and sensitivity have been achieved with liquid chromatography (LC)  techniques with mass spectrometry mass spectrometry
or mass spectroscopy
Analytic technique by which chemical substances are identified by sorting gaseous ions by mass using electric and magnetic fields. (MS) used for detection. Magni et al. (8) described such a method that allows the simultaneous identification and quantification of 4 sulfonylureas in serum by electrospray LC-M5. The technique presented by Susanto and Reinauer (9) may lack specificity because only 1 ion per compound for the selected-ion mode detection was used. Recently, Maurer et al. (10) reported a procedure for screening, identification, and quantification of several sulfonylureas in plasma. Analysis was by an atmospheric pressure chemical ionization Atmospheric pressure chemical ionization (APCI) is an ionization method used in mass spectrometry. It is a form of chemical ionization which takes place at atmospheric pressure. LC-MS equipped with a single quadrupole A quadrupole is one of a sequence of configurations of electric charge or gravitational mass that can exist in ideal form, but it is usually just part of a multipole expansion of a more complex structure reflecting various orders of complexity. . Sulfonylureas were detected by analysis in positive full-scan mode recorded at 2 fragmentor voltages, followed by library-assisted identification using a home-made LC-MS reference library.
Because ion-trap detectors measure all ions retained in the trapping steps, they do not experience sensitivity losses when run in the full-scan mode compared, for example, with triple-quadrupole MS (11). In this report, we describe an LC method coupled to ion-trap MS operated in full MS-MS scan mode to screen, identify, and quantify 8 sulfonylureas of interest in clinical toxicology.
Materials and Methods
The sulfonylureas were kindly supplied by the following manufacturers: glibenclamide, glimepiride, and tolbutamide tolbutamide /tol·bu·ta·mide/ (tol-bu´tah-mid) a sulfonylurea used as a hypoglycemic in the treatment of type 2 diabetes mellitus; the monosodium salt is used to test for insulinoma and diabetes mellitus. by Aventis (Paris, France); glipizide and chlorpropamide by Pfizer (Paris, France); gliclazide and carbutamide by Servier (Neuilly-sur-Seine, France); glibornuride by CSP (1) (Certified Systems Professional) An earlier award for successful completion of an ICCP examination in systems development. See ICCP.
(2) (Commerce Service P (Cournon, France); and glisoxepide by Bayer (Puteaux, France). All organic solvents and reagents were of analytical grade. Acetonitrile acetonitrile /ac·e·to·ni·trile/ (as?e-to-ni´tril) a colorless liquid with an etherlike odor used as an extractant, solvent, and intermediate; ingestion or inhalation yields cyanide as a metabolic product. and diethyl ether di·eth·yl ether
A pungent, volatile, highly flammable liquid derived from the distillation of ethyl alcohol with sulfuric acid and widely used as an inhalation anesthetic. Also called ethyl ether, ethyl oxide, sulfuric ether. were purchased from SDS 1. (company) SDS - Scientific Data Systems.
2. (tool) SDS - Schema Definition Set. ; methanol and formic acid formic acid or methanoic acid (mĕth'ənō`ĭk), HCO2H, a colorless, corrosive liquid with a sharp odor; it boils at 100.7°C; and solidifies at 8.4°C;. were obtained from Merck. Purified water was prepared on a Waters MilliQ purification system (Millipore).
Blank human plasma samples were supplied from the local blood bank (Etablissement Francais du Sang, Reims, France). Authentic patient plasma samples had been submitted to our laboratory for toxicology analysis.
CALIBRATION SOLUTION AND CALIBRATION CURVE
A stock solutions of each sulfonylurea was prepared in methanol at a concentration of 1 g/L and stored at 4 [degrees]C. The stock solutions were further diluted with a mixture of 1 g/L formic acid (in purified water) and acetonitrile (50:50 by volume) to give a series of working solutions used to prepare the calibrators. Calibration curves were prepared by adding 10 [micro]L of the appropriate calibrator calibrator
an instrument for dilating a tubular structure or for determining the caliber of such a structure. to 0.5 mL of human blank plasma; the final concentrations in plasma were 3.9, 7.8, 15.6, 31.25, 62.5, 125, and 250 [micro]g/L for glibenclamide; 7.8, 15.6, 31.25, 62.5, 125, 250, and 500 [micro]g/L for glipizide, gliclazide, and glibornuride; 15.6, 31.25, 62.5, 125, 250, 500, and 1000 [micro]g/L for glimepiride; 31.25, 62.5, 125, 250, 500, 1000, and 2000 [micro]g/L for chlorpropamide and carbutamide; and 78.1, 156.25, 312.5, 625, 1250, 2500, and 5000 [micro]g/L for tolbutamide.
Plasma samples (0.5 mL) were extracted with 2.5 mL of diethyl ether after addition of 25 [micro]L of an internal standard (IS) solution [10 mg/L glisoxepide in 1 g/L formic for·mic
1. Of or relating to ants.
2. Of, derived from, or containing formic acid.
[From Latin form acid-acetonitrile (50:50 by volume)] and 100 [micro]L of 1 mol/L HCI (Human Computer Interaction) Refers to the design and implementation of computer systems that people interact with. It includes desktop systems as well as embedded systems in all kinds of devices. . The mixture was vortex-mixed for 1 min, and then centrifuged at 30008 for 5 min. The organic layer was transferred to conical glass tubes and evaporated to dryness under a nitrogen stream at 40[degrees]C. Finally, the residue was dissolved in 150 [micro]L of 1 g/L formic acid-acetonitrile (50:50 by volume), and 5 [micro]L was injected on the LC column.
Instrumentation and chromatographic conditions. The LC-tandem MS (MS-MS) system consisted of a ThermoFinnigan Surveyor[R] LC system equipped with an autosampler. Compounds were detected, identified, and quantified in plasma by use of a ThermoFinnigan LCQ LCQ Longest Connected Queue
LCQ Launch Crew Quarters Advantage[R] trap ion mass spectrometer linked to a ThermoFinnigan Xcalibur[R] data system. Chromatographic separations were carried out on a Hypurity [C.sub.18] column [150 x 2.1 mm (i.d.); particle size, 5 [micro]m; ThermoHypersil-Keystone] with the temperature maintained at 30 [degrees]C. Samples were eluted with a mobile phase consisting of acetonitrile and 1 g/L formic acid in purified water (50:50 by volume), delivered at a flow rate of 0.3 mL/min. During use, the mobile phase was degassed by the integrated Surveyor series degasser. The entire flow was directed into the ionization ionization: see ion.
Process by which electrically neutral atoms or molecules are converted to electrically charged atoms or molecules (ions) by the removal or addition of negatively charged electrons. source without splitting. To optimize the MS-MS conditions and for creating library spectra, direct injection experiments were performed with a 500-[micro]L syringe connected to a pump set at a flow rate of 5 [micro]L/min. Appropriate solutions were prepared by dissolving the corresponding sulfonylureas in mobile phase to obtain a final concentration of 10 mg/L for each compound.
MS conditions. The ionization technique used was electrospray ionization (ESI (Edge Side Includes) A markup language for Web pages that enables elements of a Web page to be dynamically assembled in servers distributed throughout the Internet. ) in the positive-ion mode for glibenclamide, glipizide, gliclazide, glibornuride, glimepiride, and carbutamide and in the negative-ion mode for chlorpropamide and tolbutamide. The spray needle was set at a potential of 4 kV. The heated capillary was set at 200[degrees]C, and the stainless-steel capillary was held at a potential of 10 V. Nitrogen was used as drying and nebulizing gas: Drying gas temperature was set at 300[degrees]C, drying gas flow at 10 (arbitrary units), and nebulizing gas pressure at 240 kPa. The sheath gas flow rate of nitrogen was set at 40 (arbitrary units). The tube lens offset was set at 40 V and the electron multiplier voltage at 400 V peak to peak. Ultrapure helium (99.995%) was used in the trap as damping and collision gas (pressure of helium, [5.10.sup.-3] Torr). The instrument was set to acquire 3 microscans, and ion injection time into the trap was optimized by use of the integrated automatic gain control software.
MS conditions for detection, identification, and quantification. Sulfonylureas were detected by LC-MS-MS in full MS-MS scan mode (m/z 150-600). Full-scan MS-MS spectra were obtained by collision-induced dissociation of each molecular ion with a normalized collision energy of 50%. To generate fragment ions of the molecular ion through collision-induced dissociation, 2 analytical runs (7 and 3 alternating scan events) were carried out at m/z 272, 494, 367, 324, 491, 446, and 450, which correspond to the protonated molecular ions [[M+H].sup.+] of carbutamide, glibenclamide, glibornuride, gliclazide, glimepiride, glipizide, and glisoxepide (IS), respectively, and at m/z 275, 269, and 448, which correspond to the deprotonated molecular ions [[M-H].sup.-] of chlorpropamide, tolbutamide, and glisoxepide (IS), respectively. All full MS-MS spectra were recorded by scanning from m/z 150 to 600. Total run times were 7.5 min for positive- and 4 min for negative-ion mode analysis.
Reference MS-MS spectra of all sulfonylureas were collected individually by direct injection via the integrated syringe pump. These spectra, obtained by use of a normalized collision energy of 50%, were added to a custom full MS-MS library (including ~2000 compounds to date). Positive peaks were identified by comparing the underlying ESI mass spectra with the reference spectra in our MS-MS library.
Quantification was also performed in the full-scan MS-MS mode. Once full mass spectra of the product ions were generated, postacquisition data processing was designed to select particular ions for quantification (usually fragment ions with greater intensities). Peak-area ratios of the target ions of each sulfonylurea vs that of the IS (glisoxepide) were compared with calibration curves prepared under the same conditions. The latter were analyzed by unweighted linear regression Linear regression
A statistical technique for fitting a straight line to a set of data points. . It should be noted that for chlorpropamide, tolbutamide, and carbutamide, the ranges of the calibration curves were significantly lower than their known therapeutic ranges (Table 3). This was done mainly to allow the detection and quantification of very low drug concentrations as seen days or weeks after patients stopped their surreptitious SURREPTITIOUS. That which is done in a fraudulent stealthy manner. use. If drug concentrations in authentic samples exceeded the calibration range, samples were reanalyzed after appropriate dilution with drug-free plasma.
Quality control. Quality controls were prepared from a pool of blank human plasma with 3 different concentrations of each sulfonylurea added to give low, medium, and high concentrations (Table 1). Plasma aliquots were stored at -20[degrees]C until assayed and were renewed every 3 months.
Imprecision and recovery. Imprecision and recovery were assessed by replicate analysis of 10 (intraday) and 20 (interday) quality-control samples over the 3 concentrations of each of the 8 sulfonylureas. Imprecision (as CV) was expected to be <15% except at the limit of quantification (LOQ LOQ Limit of Quantitation
LOQ Limit Of Quantification
LOQ Loquitur (Latin: speaks)
LOQ Level of Quantification
LOQ List Of Questions
LOQ Laugh Out Quiet
LOQ Leadership Opinion Questionaire ; defined as the lowest concentration giving a signal-to-noise ratio >10:1), where 20% was acceptable. Recovery was calculated as: (mean measured concentration/added concentration) X 100. A recovery of 100 (15)% was considered acceptable, except at the LOQ, where 100 (20)% was acceptable.
Limits. To determine the limit of detection (LOD Lod (lōd), city (1994 pop. 51,200), central Israel. It is also known as Lydda. Its manufactures include paper products, chemicals, oil products, electronic equipment, processed food, and cigarettes. ; defined as the lowest concentration giving a signal-to-noise ratio >3:1), quality controls with decreasing amounts of each compound were assayed. Criteria for the LOQ were fulfilled by the lowest point of the calibration curve.
Carryover. The lack of carryover effect was assessed by alternately analyzing blank plasma samples (n = 3) and plasma samples containing concentrations at the upper LOQ of each compound (n = 3). The residual concentration found in the first blank plasma sample following a high concentration sample was used to calculate the rate of carryover. It was considered minimal if <0.5% of the LOQ.
Extraction recoveries. Extraction recoveries from human plasma were evaluated at low and high concentrations (n = 5). The samples were extracted without IS according to the procedure described above; 25 [micro]L of the IS solution was then added to the organic phase, and the sample was evaporated to dryness. The residue was dissolved in 150 [micro]L of mobile phase before analysis. For controls (n = 5), 25 [micro]L of IS was added to mixtures of the 8 sulfonylureas prepared in mobile phase at the low and high concentrations; the mixtures were then gently evaporated. The residue of each calibration mixture was then dissolved in 150 [micro]L of mobile phase and analyzed. Recoveries were calculated by comparing the peak areas of controls with those of plasma samples with added IS.
[FIGURE 1 OMITTED]
Specificity and ion suppression test suppression test A test or assay–eg, dexamethasone suppression test, used to determine whether a substance–hormone or protein being produced in excess is under the control of regulating or releasing factor(s), and therefore responsive to a feedback . We evaluated the specificity of the method by analyzing 10 different plasma samples obtained from healthy volunteers who had not received any of the sulfonylureas under investigation. The ion suppression effect for the method was also assessed with these plasma samples. After extraction, each plasma sample was injected into the LC-MS-MS system while high concentrations of the 8 drugs and IS were continuously infused post column (flow rate of 5 [micro]L/min), as described by Muller et al. (12). Because glisoxepide and chlorpropamide elute e·lute
tr.v. e·lut·ed, e·lut·ing, e·lutes
To extract (one material) from another, usually by means of a solvent.
[From Latin at the same time in the negative-ion mode, additional ion suppression experiments were carried out to check for a potential mutual ion suppression effect of these 2 compounds. Briefly, extracted samples containing a low concentration of either glisoxepide or chlorpropamide, with or without a high concentration of the corresponding compound added, were injected into the LC-MS-MS system (n = 3).
Results and Discussion
MS-MS ANALYSIS, SCREENING, AND QUANTIFICATION In contrast to Maurer et al. (10), we chose ESI over the atmospheric pressure chemical ionization mode because ESI had a higher sensitivity under our experimental conditions. Positive--or negative-ion mode was chosen to obtain the most intense signal for the molecular ion of each compound. In ESI-MS mode, the molecular cation cation (kăt'ī`ən), atom or group of atoms carrying a positive charge. The charge results because there are more protons than electrons in the cation. [[M+H].sup.+] or anion anion (ăn`ī'ən), atom or group of atoms carrying a negative charge. The charge results because there are more electrons than protons in the anion. [[M-H].sup.-] represented one of the most prominent fragments for each of the 8 compounds. Except for carbutamide, chlorpropamide, tolbutamide, and the IS when ionized in negative-ion mode, the full-scan MS spectra of compounds showed peaks at m/z M+22, which is a clear indication of the formation of sodium adducts. The mass spectra of chlorpropamide and tolbutamide gave peaks at m/z 550 and 561, indicating the presence of dimers. The protonated or deprotonated molecular ions were chosen as precursor ions for MS-MS analysis. The MS-MS spectra data for the 8 sulfonylureas obtained at a normalized collision energy of 50% are shown in Table 1. All full MS-MS spectra showed characteristic patterns, allowing unambiguous and rapid identification of the compounds by comparison with our full MS-MS reference library.
For quantification purposes, glisoxepide was chosen as an IS because this compound has not been marketed in France. However, because the presence of glisoxepide in a patient's sample can never be fully excluded, patient samples were extracted without IS added and analyzed before the final analysis. If one or several sulfonylureas were positively identified and confirmed, quantification of the corresponding compounds) was performed in the full-scan MS-MS mode. Postacquisition data processing of full-scan MS-MS data permitted the "extraction" of analytes of interest by selection of specific product ions. This mode is known to be the most sensitive MS setup for an ion-trap detector; it thus permits quantitative analysis of analytes in complex matrices with high sensitivity. Indeed, because ion traps measure all ions retained in the trapping steps, they do not experience sensitivity losses in full-scan mode as does, for example, triple-quadrupole MS (11). Moreover, full-scan MS-MS mode provides additional specificity over conventional selected- or multiple-reaction monitoring experiments without sacrificing sensitivity.
Reconstructed ion chromatograms of a blank plasma enriched with IS and therapeutic concentrations of all 8 sulfonylureas are shown in Fig. 1. In the positive-ion mode, chromatographic separation is almost complete within 8 min; therefore, in light of the absence of coeluting compounds, no interference by ion suppression, particularly in the high concentration range, is expected. In the negative-ion mode, glisoxepide (IS) and chlorpropamide almost coeluted, and total run time was shorter than 4 min per sample.
The results of the method validation in human plasma are listed in Table 2. Imprecision and recovery were within ranges defined as acceptable for bioanalytical purposes (13). Interday imprecision (as CV) ranged from 1.8% to 18%, and recovery ranged from 81.3% to 118.2%. Interday imprecision did not exceed 10% over the 3 concentrations, except for the low concentration of tolbutamide (17%). The Interday recovery of the method was satisfactory. Calibration curves were linear over the working concentration ranges with coefficients of determination ([r.sup.2]) >0.990 in all cases (Table 3). The LOD are reported in Table 3. LOQ were defined as the lowest concentration used for the calibration curves with a signal-to-noise ratio [greater than or equal to] 10 and for which the CV did not exceed 20%, and recoveries were 80%-100% (Table 3). Under our experimental conditions, the carryover effect was minimal, with carryover <0.3% of the LOQ.
Extraction recoveries from human plasma were acceptable for glibenclamide, glibornuride, gliclazide, glimepiride, and glipizide with values ranging between 68% and 87% at low concentrations and between 63% and 83% at high concentrations (Table 3). In contrast, carbutamide, chlorpropamide, and tolbutamide had low recoveries, most notably tolbutamide (<25%). Nevertheless, these recoveries were very reproducible at each concentration, and good calibration curves and coefficients of determination could be obtained. Moreover, the high sensiuvity of the MS instrument made it possible to detect and quantify these 3 compounds from subtherapeutic sub·ther·a·peu·tic
Below the dosage levels used to treat diseases: subtherapeutic feeding of penicillin to livestock.
sub to toxic concentrations (after appropriate dilution of samples if required) with good precision.
The analysis of 10 blank plasma samples from healthy volunteers showed no interfering peaks on the chromatograms. It is well known that ion suppression (which can be caused by interactions between matrix and analyte in solution when sprayed by the atmospheric pressure ionization source) is usually encountered with ESI (14); thus, this effect is usually evaluated when ESI spectrometry is used (12). In our assay, we observed no ion suppression effect in any of the blank plasma extracts at the expected retention times of the different sulfonylureas. In agreement with Muller et al. (12), we observed ion suppression effects at the LC solvent front (retention time <2 min, i.e., during the elution elution /elu·tion/ (e-loo´shun) in chemistry, separation of material by washing; the process of pulverizing substances and mixing them with water in order to separate the heavier constituents, which settle out in solution, from the of nonretained compounds), but this did not interfere with the ionization of sulfonylureas assayed in either positive--or negative-ion mode. Additionally, this shows that appropriate chromatographic separation is needed before introduction of the sample into the ion source because we found no ion suppression for the analytes of interest in their specific retention-time windows. Other ion suppression experiments showed that no reciprocal ion suppression of glisoxepide and chlorpropamide occurred during their coelution.
APPLICATION TO AUTHENTIC CLINICAL CASES
Between January 2003 and December 2004, we searched for the presence of sulfonylurea-type hypoglycemic drugs in 134 French patients who presented with unexplained and severe hypoglycemia. Using the presented analytical method, we found that 9 of these cases were likely to be related to surreputious use of sulfonylureas [4 men with a mean (SD) age of 64.0 (11.8) years and 5 women with a mean age of 63.2 (23.3) years]. Glibenclamide, glimepiride, and gliclazide were detected alone in 3, 2, and 4 patients, respectively. As shown in Table 4, plasma concentrations of glibenclamide and glimepiride were usually in the therapeutic ranges. Concerning gliclazide, in 1 case, the plasma concentrations were 2 times higher than the upper limit of the therapeutic range (i.e., 4000 [micro]g/L). Another case had a plasma concentration in the therapeutic range. Finally, plasma concentrations in the last 2 cases were well below the lower limit of the therapeutic range (i.e., 250 [micro]g/L). Because the elimination half-life of gliclazide in humans is ~20 h, such low concentrations may be attributable to a delay (several days) between ingestion ingestion /in·ges·tion/ (-chun) the taking of food, drugs, etc., into the body by mouth.
1. The act of taking food and drink into the body by the mouth.
2. and blood sampling. In conclusion, this method allows for the rapid screening and reliable identificauon of sulfonylureas in human plasma. Detection of sulfonylureas in plasma is performed by use of an ion-trap mass spectrometer with a highly selecuve LC-MS-MS procedure combined with library searching of MS-MS spectra. Quantification is achieved by use of a precise, accurate, and sensitive method developed in full-scan MS-MS mode. This assay has been successfully applied to the detection of Munchausen syndrome in authentic cases of patients with hypoglycemic crises of unknown origin.
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APCI Air Products & Chemicals, Inc.
APCI Association of Professional Color Imagers
APCI Advisory Panel on Country Information (UK)
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GUILLAUME HOIZEY, (1) * DENIS LAMIABLE, (1) THIERRY TRENQUE, (1,2) ARNAUD ROBINET, (1) LAURENT BINET, (1) MATTHIEU L. KALTENBACH, (3) SANDRINE HAVET, (2) and HERVE MILLART (1)
 Laboratoire de Pharmacologie et Toxicologie, Hopital Maison Blanche, CHU de Reims, France.
 Centre Regional de Pharmacovigilance, OHU OHU Overhead Unit (Heads-up Guidance System)
OHU Overseas Home Ported Unit de Reims, France.
 Laboratoire de Pharmacologie et de PharmacocinEtique, UFR UFR Unité de Formation et de Recherche (French universities research and teaching unit)
UFR Union of Republican Forces (Guinea)
UFR Ultrafiltration Rate (kidney dialysis) de Pharmacie, Reims, France.
 Nonstandard non·stan·dard
1. Varying from or not adhering to the standard: nonstandard lengths of board.
2. abbreviations: LC, liquid chromatography; MS-MS, tandem mass spectrometry Tandem mass spectrometry, also known as MS/MS, involves multiple steps of mass spectrometry selection, with some form of fragmentation occurring in between the stages. ; IS, internal standard; ESI, electrospray ionization; LOQ, limits) of quantification; and LOD, limits) of detection.
* Address correspondence to this author at: Laboratoire de Pharmacologie et Toxicologie, Hopital Maison Blanche, CHU de Reims, 45, rue Cognacq-Jay, 51092 Reims cedex, France. Fax 33-3-2678-8456; e-mail email@example.com.
Received March 9, 2005; accepted June 13, 2005.
Previously published online at DOI (Digital Object Identifier) A method of applying a persistent name to documents, publications and other resources on the Internet rather than using a URL, which can change over time. : 10.1373/clinchem.2005.050864
Table 1. ESI-MS-MS spectral data for the 8 sulfonylureas and glisoxepide (IS). Sulfonylurea Parent ion, m/z Daughter ions, (a) m/z Glibenclamide 494 [[M + H].sup.+] 369 (100), 395 (5) Glipizide 446 [[M + H].sup.+] 321 (100), 347 (24), 286 (3), 304 (2) Gliclazide 324 [[M + H].sup.+] 127 (100), 110 (43), 168 (31), 153 (18), 151 (8), 128 (7) Glibornuride 367 [[M + H].sup.+] 349 (100), 170 (58), 196 (40), 152 (31) Glimepiride 491 [[M + H].sup.+] 352 (100) Carbutamide 272 [[M + H].sup.+] 156 (100), 173 (34), 229 (14), 155 (3) Chlorpropamide 275 [[M + H].sup.+] 190 (100) Tolbutamide 269 [[M + H].sup.+] 170 (100) Glisoxepide 450 [[M + H].sup.+] 310 (100), 141 (99), 311 (14), 350 (8) Glisoxepide 448 [[M + H].sup.+] 308 (100), 225 (20) (a) Values in parentheses are the relative intensities. Table 2. Intraday (n = 10) and interday (n = 20) precision and recovery. Mean measured, [micro]g/L Added, Analyte [micro]g/L Intraday Interday Carbutamide Low 31.2 25.5 27.3 Medium 250 233 235 High 1000 984 1072 Chlorpropamide Low 31.2 36.9 36.4 Medium 250 243 281 High 1000 1051 1055 Glibenclamide Low 3.91 3.72 3.77 Medium 31.25 30.8 31.9 High 125 127.3 127 Glibornuride Low 7.81 6.56 7.17 Medium 62.5 59.9 58 High 250 242.1 246 Gliclazide Low 7.81 8.15 7.98 Medium 62.5 64.7 69 High 250 273.8 266 Glimepiride Low 15.6 15.3 17.2 Medium 125 118 116 High 500 483.8 468 Glipizide Low 7.81 6.62 7.37 Medium 62.5 54.5 69 High 250 253.8 241 Tolbutamide Low 78.1 63.5 72.6 Medium 625 604 615 High 2500 2540 2468 CV, % Recovery, (a) % Analyte Intraday Interday Intraday Interday Carbutamide Low 16.0 9.6 81.5 87.4 Medium 5.6 6.1 93.2 93.9 High 7.0 8.6 98.4 107.2 Chlorpropamide Low 6.8 8.8 118.2 116.6 Medium 3.4 8.5 97.0 112.6 High 2.6 5.2 105.1 105.5 Glibenclamide Low 18 9.8 95.1 96.5 Medium 7.2 7.6 98.4 101.9 High 4.7 4.8 101.8 101.9 Glibornuride Low 12 9.4 83 91.8 Medium 4.4 8.3 95.9 92.7 High 6.6 5.9 96.8 98.4 Gliclazide Low 7.4 6.9 104.3 102.2 Medium 5.5 6.9 103.6 110.9 High 6.2 5.5 109.5 106.5 Glimepiride Low 12 9.8 98.2 109.9 Medium 8.6 9.2 94.6 92.9 High 10 6.4 96.8 93.7 Glipizide Low 7.4 9.9 84.8 94.3 Medium 2.3 6.9 87.3 110.4 High 5.4 7.2 101.5 96.5 Tolbutamide Low 8.6 17 81.3 92.9 Medium 7.1 8.2 96.7 98.4 High 1.8 7.5 101.6 98.7 (a) Expressed as (mean measured concentration/added concentration) x 100%. Table 3. LOD, LOQ, therapeutic concentrations, linearity, and extraction recoveries of sulfonylureas in human plasma. Therapeutic LOD, LOQ, concentrations, Analyte [micro]g/L [micro]g/L [micro]g/L Carbutamide 1.98 31.25 <20 000 Chlorpropamide 1.98 31.25 <30 000 Glibenclamide 0.24 3.91 30-200 Glibornuride 1.95 7.81 25-50 Gliclazide 0.49 7.81 250-4000 Glimepiride 0.98 15.6 <300 Glipizide 1.95 7.81 100-1000 Tolbutamide 4.90 78.1 <20 000 Coefficient of Extraction recovery Linearity, determination Analyte [micro]g/L ([r.sup.2]) [micro]g/L % Carbutamide 31.25-2000 0.999 31.25 37 1000 44 Chlorpropamide 31.25-2000 0.990 31.25 36 1000 33 Glibenclamide 3.91-250 0.997 3.91 87 125 76 Glibornuride 1.81-500 0.999 7.81 81 250 79 Gliclazide 7.81-500 0.998 7.81 78 250 83 Glimepiride 15.6-1000 0.999 15.6 86 500 70 Glipizide 7.81-500 0.998 7.81 68 250 63 Tolbutamide 78.1-5000 0.996 78.1 21 2500 25 Table 4. Plasma sulfonylurea concentrations in authentic cases of surreptitious use of sulfonylureas. Concentration, Case Hypoglycemic agent [micro]g/L 1 Glibenclamide 24 2 Glibenclamide 28 3 Glibenclamide 88 4 Glimepiride 311 5 Glimepiride 550 6 Gliclazide 9080 7 Gliclazide 1283 8 Gliclazide 56 9 Gliclazide 23