Validation and implementation of a liquid chromatography/tandem mass spectrometry assay to quantitate aminoflavone (NSC 686288) in human plasma

A reverse-phase liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) method was developed and validated for determination of aminoflavone (AF) in human plasma. Sample preparation involved a liquid–liquid extraction by the addition of 0.25 mL of plasma with 1.0 mL ethyl acetate containing 50 ng/mL of the internal standard zileuton. The analytes were separated on a Waters X-Terra? MS C₁₈ column using a mobile phase consisting of methanol/water containing 0.45% formic acid (70:30, v/v) and isocratic flow at 0.2 mL/min for 6 min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the AF concentration range of 5–2000 ng/mL in human plasma. The lower limit of quantitation (LLOQ) was 5 ng/mL for AF in human plasma. The accuracy and within- and between-day precisions were within the generally accepted criteria for bioanalytical method (<15%). This method was successfully applied to characterize AF plasma concentration-time profile in the cancer patients in a phase I trial.     

Validation and implementation of a liquid chromatography/tandem mass spectrometry assay to quantitate dimethyl benzoylphenylurea (BPU) and its five metabolites in human plasma and urine for clinical pharmacology studies

A method has been developed for the quantitation of N-[4-(5-bromo-2-pyrimidinyloxy)-3-methylphenyl]-N'-(2-dimethylamino-benzoyl)urea (BPU) and its metabolites in human plasma and urine. BPU and metabolites were separated on a C18 column with acetonitrile-water mobile phase containing 0.1% formic acid using isocratic flow for 5 min. The analytes were monitored by tandem mass spectrometry. Calibration curves were generated over the range of 2.5-500 ng/mL for BPU, mmBPU, and aminoBPU in plasma; and 0.1-20, 0.1-20, 0.5-100, 10-2000, 1-200, and 3-600 ng/mL for BPU, mmBPU, aminoBPU, G280, G308, and G322 in urine, respectively. The method has been successfully applied to study the pharmacokinetics of BPU.     

Validation and implementation of a method for determination of bryostatin 1 in human plasma by using liquid chromatography/tandem mass spectrometry

A new sensitive and specific method using liquid chromatography/tandem mass spectrometry for determination of bryostatin 1 was developed and validated. Sample pretreatment involved a double liquid-liquid extraction step with a mixture of acetonitrile/n-butyl chloride (1/4, v/v). Separation of the compound of interest, including the internal standard paclitaxel, was achieved on a Waters X-Terra C18 (50 x 2.1 mm i.d., 3.5 microm) analytical column with acetonitrile/water mobile phase (80:20, v/v) containing 0.1% formic acid using isocratic flow at 0.15 mL/min for 13 min. The analytes of interest were monitored by tandem mass spectrometry with electrospray positive ionization. The linear calibration curves were generated over the range of 50-2000 pg/mL with values for the coefficient of determination of >0.99. The values for both within-day and between-day precision and accuracy were <15%. This method was used to characterize the plasma pharmacokinetics of bryostatin 1 at doses of 20 microg/m2) to optimize treatment with this agent.     

Validation and application of a sensitive assay for butorphanol in human plasma by high-performance liquid chromatography with tandem mass spectrometry detection

A sensitive and convenient high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay for the opioid receptor agonist-antagonist butorphanol in human plasma is described. BC-2605, a cyclopropyl analogue of butorphanol, was employed as an internal standard. Butorphanol was recovered from plasma (84.4 +/- 10.9%) by liquid-liquid extraction. The mobile phase flow-rate was 0.3 ml/min and consisted of methanol-water-formic acid (90:10:0.1, v/v/v). The analytical column (4.6 x 100 mm) was packed with Partisil C(8) (5 microm). The standard curve was linear from 13.7 to 1374 pg/ml (r(2)>0.99). The lower limit of quantitation was 13.7 pg/ml. The assay was specific, accurate (% deviation from nominal concentrations were <15%), precise and reproducible (within- and between-day coefficients of variation <7%). Butorphanol in plasma was stable over 3 freeze/thaw cycles and at room temperature for 1 day. The utility of the assay was demonstrated by following butorphanol plasma concentrations in two healthy subjects for 24 h following a 1 mg intranasal dose.     

Determination of sugammadex in human plasma, urine, and dialysate using a high-performance liquid chromatography/tandem mass spectrometry assay

Sugammadex (Bridion?, Merck Sharp & Dohme Corp., Oss, The Netherlands) is a modified γ-cyclodextrin which has the ability to reverse the neuromuscular blockade induced by the steroidal neuromuscular blocking agents rocuronium and vecuronium. The objective of the current study is to describe the bioanalytical methods that have been developed and validated according to US Food and Drug Administration guidelines on bioanalytical method validation, and subsequently applied to determine total sugammadex (i.e., free sugammadex plus sugammadex bound to the neuromuscular blocking agent) in human heparinized plasma, urine and dialysate. Sugammadex was extracted from human plasma and urine using solid phase extraction with Isolute HAX 96-well extraction plates; no extraction was performed on dialysate samples. Samples from plasma, urine, and dialysate were analyzed on a Polaris? C18-A PEEK (polyaryletheretherketone) analytical column (50 mm × 4.6 mm internal diameter, 5 μm) with a linear mobile phase gradient of 0.1% (v/v) formic acid in water:methanol from 70:30 to 20:80. The flow rate was 1 mL/min with a total run time for each injection of 6 min. Tandem mass spectrometric detection was conducted using multiple reaction monitoring under negative ion mode with a turbo ion-spray interface to quantify the concentration of sugammadex. Inter- and intra-assay precision and accuracy were within pre-defined acceptance limits. The presence of rocuronium did not interfere with the assay in plasma, urine or dialysate; similarly, vecuronium did not interfere with the plasma assay (not tested for interference in urine or dialysate). Sugammadex was found to be stable in plasma, urine and dialysate in the short-term at room temperature, in the long-term at -20°C, and after several freeze/thaw cycles. The validated bioanalytical methods developed here have been successfully applied in a series of clinical studies for the determination of total sugammadex in plasma, urine and dialysate.     

Determination of azatadine in human plasma by liquid chromatography/tandem mass spectrometry

A sensitive method using liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) was developed and validated for the analysis of antihistamine drug azatadine in human plasma. Loratadine was used as internal standard (IS). Analytes were extracted from human plasma by liquid/liquid extraction using ethyl acetate. The organic phase was reduced to dryness under a stream of nitrogen at 30 °C and the residue was reconstituted with the mobile phase. 5 μL of the resulting solution was injected onto the LC-MS/MS system. A 4.6 mm × 150 mm, I.D. 5 μm, Agilent TC-C(18) column was used to perform the chromatographic analysis. The mobile phase consisted of ammonium formate buffer 0.010 M (adjusted to pH 4.3 with 1M formic acid)/acetonitrile (20:80, v/v) The chromatographic run time was 5 min per injection and flow rate was 0.6 mL/min. The retention time was 2.4 and 4.4 min for azatadine and IS, respectively. The tandem mass spectrometric detection mode was achieved with electrospray ionization (ESI) iron source and the multiple reaction monitoring (MRM) (291.3 → 248.2m/z for azatadine, 383.3 → 337.3m/z for IS) was operated in positive ion modes. The low limit of quantitation (LLOQ) was 0.05 ng/mL. The intra-day and inter-day precision of the quality control (QC) samples was 8.93-11.57% relative standard deviation (RSD). The inter-day accuracy of the QC samples was 96.83-105.07% of the nominal values.     

Quantitation of bergenin in human plasma by liquid chromatography/tandem mass spectrometry

This paper reports the development and validation of an assay for quantitation of bergenin in human plasma using liquid chromatography/tandem mass spectrometry (LC–MS/MS). Bergenin and the internal standard (I.S.), 5-bromo-2,4(1H,3H)-pyrimidinedione (5-BrU), were separated by reversed phase HPLC and quantitated by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM) in the negative ion mode. The most intense [M?H]^? MRM transition of bergenin at m/z 326.9 → 312.3 was used for quantitation and the transition at m/z 188.9 → 42.2 was used to monitor 5-BrU. Stability issues with bergenin required the addition of ascorbic acid to plasma samples prior to storage and analysis within 10 days storage at ?80 °C. The method was linear in the range 3–1000 ng/mL with intra- and inter-day precision of 3.94–5.96 and 1.62–8.31%, respectively, and accuracy <2.33%. The assay was successfully applied to a pharmacokinetic study in healthy volunteers after administration of a single 250 mg oral dose.     

Validation and application of an assay for the determination of mevalonic acid in human plasma by liquid chromatography tandem mass spectrometry

The validation of a method for the determination of mevalonic acid (MVA; after conversion to the lactone, MVAL) in human plasma, using high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS-MS), is reported. MVAL and deuterated internal standard were extracted from human plasma samples using automated solid-phase extraction. Analysis was conducted by column-switching, reversed-phase LC-MS-MS, using two hyper-cross-linked styrene-divinylbenzene copolymer sorbent reversed-phase columns. An assay range of 0.2-35 ng/ml and a lower limit of quantitation (LLOQ) of 0.2 ng/ml were achieved with acceptable accuracy and precision. MVA was stable in plasma under a variety of storage conditions.     

Validation of a quantitative assay for human neutrophil peptide-1, -2, and -3 in human plasma and serum by liquid chromatography coupled to tandem mass spectrometry

A quantitative assay for simultaneous measurement of individual human neutrophil peptide-1, -2 and -3 concentrations will aid in exploring the potential of these antimicrobial peptides as biomarkers for various diseases. Therefore, a liquid chromatography–tandem mass spectrometry method has been developed and validated to allow separate quantification of the three human neutrophil peptides in human plasma and serum. Plasma and serum samples (100 μl) were deproteinized by precipitation, followed by chromatographic separation on a Symmetry 300 C₁₈ column (50 mm × 2.1 mm I.D., particle size 3.5 μm), using a water–methanol gradient containing 0.25% (v/v) formic acid and human alpha-defensin 5 as internal standard. Tandem mass spectrometric detection was performed on a triple quadrupole mass spectrometer equipped with electrospray ionization. Despite low fragmentation efficiency of the antimicrobial peptides, multiple reaction monitoring was used for detection, though selecting the quaternary charged ions as both precursor and product. The method was linear for concentrations between 5 and 1000 ng/ml with a limit of detection around 3 ng/ml for all peptides. Intra- and inter-assay precisions were 14.8 and 19.1%, respectively, at the lowest measured endogenous concentration (6.4 ng/ml of HNP-1 in plasma), representing the lower limit of quantification of the assay. Recoveries of HNP-1, -2 and -3 from plasma and serum ranged between 85 and 128%. Analysis of serum samples from intensive care patients showed average concentrations of 362, 570 and 143 ng/ml for HNP-1, -2 and -3, respectively.     

A rapid and sensitive method for determination of sorafenib in human plasma using a liquid chromatography/tandem mass spectrometry assay

A rapid, sensitive and specific method was developed and validated using LC/MS/MS for determination of sorafenib in human plasma. Sample preparation involved a single protein precipitation step by the addition of 0.1 mL of plasma with 0.5 mL acetonitrile. Analysis of the compounds of interest including the internal standard ([(2)H(3)(15)N] sorafenib) was achieved on a Waters X-Terra C(18) (150 mm x 2.1mm i.d., 3.5 microm) analytical column using a mobile phase consisting of acetonitrile/10 mM ammonium acetate (65:35, v/v) containing 0.1% formic acid and isocratic flow at 0.2 mL/min for 6 min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 7.3-7260 ng/mL for the human plasma samples with values for the coefficient of determination of >0.96. The values for both within day and between day precision and accuracy were well within the generally accepted criteria for analytical methods (<15%).     

Quantitative analysis of racecadotril metabolite in human plasma using a liquid chromatography/tandem mass spectrometry

Orally administered racecadotril is rapidly hydrolyzed to the more potent enkephalinase inhibitor thiorphan in vivo. A sensitive and specific liquid chromatography/tandem mass spectrometry method was developed and validated to quantify thiorphan in human plasma using lisinopril as the internal standard. After a simple protein precipitation with methanol, the post-treatment samples were analyzed on a CN column interfaced with a triple-quadruple tandem mass spectrometer using negative electrospray ionization. The method was validated to demonstrate the specificity, lower limit of quantification, accuracy, and precision of measurements. The assay was linear over the concentration range 9.38-600 ng/mL using a 5 microL aliquot of plasma. The correlation coefficients for the calibration curves ranged from 0.9985 to 0.9995. The intra- and inter-day precisions over the entire concentration were not more than 6.33%. Methanol and water (35:65, v/v) is used as the isocratic mobile phase, with 0.1% of formic acid in water. The method was successfully applied for pharmacokinetic study after a single oral administration of 200 mg racecadotril to 20 healthy volunteers.     

Two-dimensional liquid chromatography/tandem mass spectrometry analysis of Gradiflow fractionated native human plasma

One of the major challenges facing protein analysis is the dynamic range of protein expression within massively complex samples (Corthals, G. L. et al.., Electrophoresis 2000, 21, 1104-1115). In plasma this difference is as great as ten orders of magnitude, and this is currently beyond the range of detection achievable by any of the analytical techniques. Plasma has the additional challenge of having a few highly abundant proteins, such as albumin, which mask the detection of lower abundance and biologically significant proteins. The use of the Gradiflow BF400 as a fractionation tool to deplete highly abundant albumin from human plasma is reported here. A sequential three-step protocol was performed on five plasma samples as part of the International Plasma Proteome Project organised by the HUPO; four containing different anticoagulants: EDTA, citrate, heparin and a control sample (NIBSC); and a serum sample. Plasma from an alternate source also underwent fractionation and served as an in-house control. Time modulation between 1 and 7 h was observed for the depletion of albumin from these samples. Following albumin depletion, each fraction was trypsin-digested and the peptides were fractionated further using a 2-D LC-MS/MS. Differences in the total number of proteins identified for each sample were also noted.     

Felodipine quantification in human plasma by high-performance liquid chromatography coupled to tandem mass spectrometry

A rapid, sensitive, robust and specific method was developed for the determination and quantitation of felodipine, in human blood plasma by liquid chromatography coupled with tandem mass spectrometry using nimodipine as internal standard. Felodipine was extracted from 0.5 mL human plasma by use of a liquid/liquid procedure using diethyl ether/hexane (80/20, v/v) as eluent. The method included a chromatographic run of 5 min using a C(18) analytical column (100 mm x 4.6 mm i.d.) and the calibration curve was linear over the range from 0.02 to 10 ng mL(-1) (r(2) > 0.994). The between-run precision, determined as relative standard deviation of replicate quality controls, was 5.7% (0.06 ng mL(-1)), 7.1% (0.6 ng mL(-1)) and 6.8% (7.5 ng mL(-1)). The between-run accuracy was +/- 0.0, 2.1 and 3.1% for the above-mentioned concentrations, respectively.     

Simultaneous quantification of carbamate insecticides in human plasma by liquid chromatography/tandem mass spectrometry

Carbofuran (CFN), carbosulfan (CSN) and fenobucarb (FBC) are carbamate pesticides that are widely used in gardening and agriculture for the control of insects. Human poisoning due to occupational or self-poisoning exposures is also reported, so assays are required to quantify the plasma concentration of these insecticides. An LC-MS/MS method was developed and validated for the simultaneous quantification of these three carbamate insecticides in the plasma of patients with acute intentional self-poisoning. Plasma samples were pretreated by acetonitrile for protein precipitation. Chromatography was carried out on a Luna C18(2) analytical column with gradient elution using a mobile phase containing acetonitrile and water with 10mM ammonium acetate. Mass spectrometric analysis was performed by an Applied Biosystems MDS Sciex API 2000 triple quadrupole mass spectrometer coupled with electrospray ionization (ESI) source in the positive ion mode. The total run time was 7 min. The assay was validated over a concentration range from 10 to 1000 ng/ml for CSN and FBC and 20-2000 ng/ml for CFN. The precision and accuracy for both intra- and inter-day determination of all analytes were acceptable (<15%). No significant matrix effect was observed. Stability of compounds was established for short term bench and autosampler storage as well as freeze/thaw cycles. The method was effectively applied to 270 clinical samples from patients with a history of acute intentional carbamate self-poisoning.     

Enantiomeric determination of amlodipine in human plasma by liquid chromatography coupled to tandem mass spectrometry

A sensitive method for the separation and determination of amlodipine enantiomers in plasma has been developed based on solid-phase extraction (SPE) with disposable extraction cartridges (DECs) in combination with chiral liquid chromatography (LC). The SPE technique is used to isolate the drug from the biological matrix and to prepare a cleaner sample before injection and analysis by HPLC coupled to mass spectrometry. The DEC is filled with ethyl silica (50 mg) and is first conditioned with a 2.5% ammonia in methanol solution and then with ammonium acetate buffer. A 1.0-ml volume of plasma is then applied on the DEC. The washing step is first performed with ammonium acetate buffer and secondly with a mixture of water and methanol (65:35, v/v), while the final elution step is obtained by dispensing methanol containing 2.5% of ammonia. The eluate is then collected and evaporated to dryness before being dissolved in the LC mobile phase and injected into the LC system. The stereoselective analysis of amlodipine is achieved on a Chiral AGP column containing alpha(1)-acid glycoprotein as chiral selector by using a mobile phase consisting of a 10-mM acetate buffer (pH 4.5) and 1-propanol (99:1, v/v). The LC system is coupled to tandem mass spectrometry with an APCI interface in the positive-ion mode. The chromatographed analytes are detected in the selected reaction monitoring mode (SRM). The MS/MS ion transitions monitored are 409 to 238 for amlodipine, and 260 to 116 for S-(-)-propranolol used as internal standard (IS). The method was validated considering different parameters, such as linearity, precision and accuracy. The limit of quantitation was found to be 0.1 ng/ml for each amlodipine enantiomer.     

Determination of misoprostol acid in human plasma by liquid chromatography coupled to tandem mass spectrometry

A rapid and sensitive liquid chromatographic/tandem mass spectrometric method for determination of misoprostol acid, the active metabolite of misoprostol, was developed and validated. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a C(18) column. An API 4000 tandem mass spectrometer equipped with Turbo IonSpray ionization source was used as detector and was operated in the negative ion mode. Multiple reaction monitoring using the precursor to product ion combinations of m/z 367-249 and 296-269 was performed to quantify misoprostol acid and the internal standard hydrochlorothiazide, respectively. The method was linear in the concentration range of 10.0-3000 pg mL(-1) using 200 microL plasma. The lower limit of quantification was 10.0 pg mL(-1). The intra- and inter-day relative standard deviation over the entire concentration range was less than 8.3%. Accuracy determined at three concentrations (25.0, 200 and 2700 pg mL(-1) for misoprostol acid) ranged from -0.5 to 1.2% in terms of relative error. Each plasma sample was chromatographed within 3.5 min. The method was successfully used in a pharmacokinetic study of misoprostol in human plasma after an oral administration of 0.6 mg misoprostol.     

Microanalysis of N-linked oligosaccharides in a glycoprotein by capillary liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry

We have studied rapid and simple sugar mapping using liquid chromatography/electrospray ionization mass spectrometry (LC/MS) equipped with a graphitized carbon column. The oligosaccharide mixture was separated on the basis of the sequence, branching structure, and linkage, and each oligosaccharide was characterized based on its molecular mass. In this study we demonstrated the usefulness of capillary LC/MS (CapLC/MS) and capillary liquid chromatography/tandem mass spectrometry (CapLC/MS/MS) as sensitive means for accomplishing the structural analysis of oligosaccharides in a low-abundance glycoprotein. The carbohydrate heterogeneity and molecular mass information of each oligosaccharide can be readily obtained from CapLC/MS of a small amount of glycoprotein. CapLC/MS/MS provided b-ion series, which is informative with regard to monosaccharide sequence. Exoglycosidase digestion followed by CapLC/MS elucidated a carbohydrate residue linkage. Using this method, we characterized N-linked oligosaccharides in hepatocyte growth factor produced in mouse myeloma NS0 cells as the complex-type bi-, tri-, and tetraantennary terminated with N-glycolylneuraminic acids and alpha-linked galactose residues. Sugar mapping with CapLC/MS and CapLC/MS/MS is useful for monitoring glycosylation patterns and for structural analysis of carbohydrates in a low-abundance glycoprotein and thus will become a powerful tool in biological, pharmaceutical, and clinical studies.     

A new liquid chromatography/tandem mass spectrometry method for quantification of gangliosides in human plasma

Gangliosides are a family of glycosphingolipids characterized by mono- or polysialic acid-containing oligosaccharides linked through 1,3- and 1,4-β glycosidic bonds with subtle differences in structure that are abundantly present in the central nervous systems of many living organisms. Their cellular surface expression and physiological malfunction are believed to be pathologically implicated in considerable neurological disorders, including Alzheimer and Parkinson diseases. Recently, studies have tentatively elucidated that mental retardation or physical stagnation deteriorates as the physiological profile of gangliosides becomes progressively and distinctively abnormal during the development of these typical neurodegenerative syndromes. In this work, a reverse-phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay using standard addition calibration for determination of GM2, GM3, GD2, and GD3 in human plasma has been developed and validated. The analytes and internal standard were extracted from human plasma using a simple protein precipitation procedure. Then the samples were analyzed by reverse-phase ultra-performance liquid chromatography (UPLC)/MS/MS interfaced to mass spectrometry with electrospray ionization using a multiple reaction monitoring mode to obtain superior sensitivity and specificity. This assay was validated for extraction recovery, calibration linearity, precision, and accuracy. Our quick and sensitive method can be applied to monitor ganglioside levels in plasma from normal people and neurodegenerative patients.     

Simultaneous quantitation of dexamethasone palmitate and dexamethasone in human plasma by liquid chromatography/tandem mass spectrometry

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for simultaneous quantitation of dexamethasone palmitate and dexamethasone in human plasma was developed. After sample preparation by protein precipitation and liquid-liquid extraction, the analytes and internal standard (IS) were separated on a Venusil XBP-C8 column using gradient elution. Multiple reaction monitoring of dexamethasone palmitate, dexamethasone and IS used the precursor to product ion transitions at m/z 631.8-->373.1, m/z 393.2-->147.1 and m/z 264.2-->58.1, respectively. The method was linear over the ranges 1.5-1000ng/mL for dexamethasone palmitate and 2.5-250ng/mL for dexamethasone with intra- and inter-day precisions of <10% and accuracies of 100+/-7%. The assay was applied to a clinical pharmacokinetic study involving the injection of dexamethasone palmitate to healthy volunteers.     

Analysis of carbohydrate heterogeneity in a glycoprotein using liquid chromatography/mass spectrometry and liquid chromatography with tandem mass spectrometry

High-performance liquid chromatography with on-line electrospray ionization mass spectrometry (ESI-LC/MS) was investigated for the analysis of carbohydrate heterogeneity using RNase B as a model glycoprotein. Oligosaccharides released from RNase B with endoglycosidase H were reduced and separated on a graphitized carbon column (GCC). GCC-HPLC/MS in the positive-ion mode was successful in the identification of one Man5GlcNAc, three Man6GlcNAc, three Man7GlcNAc, three Man8GlcNAc, one Man9GlcNAc, and an oligosaccharide having six hexose units (Hex) and two N-acetylhexosamine units (HexNAc). The branch structures of the three Man7GlcNAc isomers were determined by liquid chromatography with tandem mass spectrometry (LC/MS/MS). LC/MS/MS analysis was shown to be useful for the detection and identification of a trace amount of Hex6HexNAc2 alditol as a hybrid-type oligosaccharide. Its structure was confirmed by the combination of LC/MS with enzymatic digestion using beta-galactosidase and N-acetyl-beta-glucosaminidase. The relative quantities of high-mannose-type oligosaccharides in RNase B detected by ESI-LC/MS are in reasonable agreement with those by UV, high-pH anion-exchange chromatography with pulsed amperometric detection, fluorophore-assisted carbohydrate electrophoresis. Our results indicate that LC/MS and LC/MS/MS can be utilized to elucidate the distribution of oligosaccharides and their structures, which differ in molecular weight, sugar sequence, and branch structure.