Journal of Chromatography B (v.878, #9-10)

A novel validated liquid chromatography–tandem mass spectrometry (LC–MS/MS) procedure was developed and fully validated for the simultaneous determination of nicotine-N-β-d-glucuronide, cotinine-N-oxide, trans-3-hydroxycotinine, norcotinine, trans-nicotine-1′-oxide, cotinine, nornicotine, nicotine, anatabine, anabasine and cotinine-N-β-d-glucuronide in human plasma or urine. Target analytes and corresponding deuterated internal standards were extracted by solid-phase extraction and analyzed by LC–MS/MS with electrospray ionization (ESI) using multiple reaction monitoring (MRM) data acquisition. Calibration curves were linear over the selected concentration ranges for each analyte, with calculated coefficients of determination (R 2) of greater than 0.99. The total extraction recovery (%) was concentration dependent and ranged between 52–88% in plasma and 51–118% in urine. The limits of quantification for all analytes in plasma and urine were 1.0 ng/mL and 2.5 ng/mL, respectively, with the exception of cotinine-N-β-d-glucuronide, which was 50 ng/mL. Intra-day and inter-day imprecision were ≤14% and ≤17%, respectively. Matrix effect (%) was sufficiently minimized to ≤19% for both matrices using the described sample preparation and extraction methods. The target analytes were stable in both matrices for at least 3 freeze–thaw cycles, 24 h at room temperature, 24 h in the refrigerator (4 °C) and 1 week in the freezer (−20 °C). Reconstituted plasma and urine extracts were stable for at least 72 h storage in the liquid chromatography autosampler at 4 °C. The plasma procedure has been successfully applied in the quantitative determination of selected analytes in samples collected from nicotine-abstinent human participants as part of a pharmacokinetic study investigating biomarkers of nicotine use in plasma following controlled low dose (7 mg) transdermal nicotine delivery. Nicotine, cotinine, trans-3-hydroxycotinine and trans-nicotine-1′-oxide were detected in the particular sample presented herein. The urine procedure has been used to facilitate the monitoring of unauthorized tobacco use by clinical study participants at the time of physical examination (before enrollment) and on the pharmacokinetic study day.
Keywords: Nicotine and nicotine metabolites; Plasma/urine; LC–ESI–MS/MS; 7-mg transdermal nicotine patch;

Simple liquid chromatography method for the quantification of irinotecan and SN38 in sheep plasma: Application to in vivo pharmacokinetics after pulmonary artery chemoembolization using drug eluting beads by Minh-Tâm Baylatry; Anne-Christine Joly; Jean-Pierre Pelage; Leila Bengrine-Lefevre; Jean-Louis Prugnaud; Alex Laurent; Christine Fernandez (738-742).
A rapid and simple liquid chromatography–fluorescence detection (LC–FD) method was developed and validated for the simultaneous quantification of irinotecan (CPT11) and SN38 in sheep plasma. Camptothecin (CPT) was used as the internal standard. A single step protein precipitation with acetonitrile was used for sample preparation. The separation was achieved using a 5 μm C18 column (250 mm × 4.5 mm, 5 μm) with a mobile phase composed of 36 mM sodium dihydrogen phosphate dehydrate and 4 mM sodium 1 heptane sulfonate–acetonitrile (72:28), the pH of the mobile phase was adjusted to 3. The flow rate was 1.45 mL/min and the fluorescence detection was operated at 355/515 nm (excitation/emission wavelengths). The run time was 13 min. The method was validated with respect to selectivity, extraction recovery, linearity, intra- and inter-day precision and accuracy, limit of quantification and stability. The method has a limit of quantification of 5 ng/mL for both CPT11 and SN38. The assay was linear over concentrations ranging from 5 to 5000 ng/mL and to 240 ng/mL for CPT11 and SN38, respectively. This method was used successfully to perform plasma pharmacokinetic studies of CPT11 after pulmonary artery embolization (PACE) in a sheep model. It was also validated for CPT11 and SN38 analysis in sheep lymph and human plasma.
Keywords: Irinotecan; SN38; HPLC; Pharmacokinetics; Sheep; Chemoembolization;

A specific, sensitive and rapid method based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) was developed for the simultaneous determination of olmesartan (OLM) and hydrochlorothiazide (HCTZ) in human plasma and urine. Solid-phase extraction (SPE) was used to isolate the analytes from biological matrices followed by injection of the extracts onto a C18 column with isocratic elution. Detection was carried out on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode using negative electrospray ionization (ESI). The method was validated over the concentration range of 1.00–1000 ng/mL and 5.00–5000 ng/mL for OLM in human plasma and urine as well as 0.500–200 ng/mL and 25.0–25,000 ng/mL for HCTZ in human plasma and urine, respectively. Inter- and intra-run precision of OLM and HCTZ were less than 15% and the accuracy was within 85–115% for both plasma and urine. The average extraction recoveries were 96.6% and 92.7% for OLM, and 87.2% and 72.1% for HCTZ in human plasma and urine, respectively. The linearity, recovery, matrix effect and stability were validated for OLM/HCTZ in human plasma and urine.
Keywords: Simultaneous; Determination; Olmesartan; Hydrochlorothiazide; HPLC-MS/MS; Human plasma and urine;

The peptide described in this report (MW 1180 Da; 10-amino acid synthetic peptide) is a potent and selective antagonist of the human B1 receptor (B1) that has been investigated for the treatment of chronic pain. A method to quantitate this peptide in human plasma has been developed to support human clinical trials designed to evaluate the safety, pharmacokinetics, and efficacy of this compound. Plasma samples (0.2 mL) were extracted using a Waters Oasis MAX (10 mg) 96-well plate and the resulting samples were analyzed using an Applied Biosystems API-5000 HPLC-MS/MS with an electrospray ionization (ESI) source. The method was validated for the determination of the B1 peptide in human plasma over the concentration range of 1–50 ng/mL. Isotopically labeled B1 peptide (13C615N2-B1 peptide) was used as an internal standard. Interday precision and accuracy, determined from analysis of quality control (QC) samples, yielded coefficients of variation (CV) of less than 5.3% and accuracy within a 2.4%. Within batch precision and accuracy determinations provided CV values of less than 7.3% and accuracy within a 6.0% bias. Precautions had to be taken to prevent B1 peptide loss to container surfaces and contamination of the HPLC-MS/MS. The validated assay was used in support of human clinical trials.
Keywords: HPLC-MS/MS; SPE; Peptide; 96-well SPE; Absorption;

Determination of melamine and cyanuric acid in human urine by a liquid chromatography tandem mass spectrometry by Mengqi Zhang; Shuijun Li; Chengyin Yu; Gangyi Liu; Jingying Jia; Chuan Lu; Jian He; Yinghua Ma; Jianmin Zhu; Chen Yu (758-762).
Melamine was found to be the etiological factor for the urinary stones epidemic in infants and young children in China in 2008. Urine level of melamine and its analog cyanuric acid may be useful markers for the evaluation of toxic effects. Liquid chromatography tandem mass spectrometry methods for the individual determination of melamine and cyanuric acid in human urine are described. Using isotope labeled internal standards during liquid–liquid extraction, the method was fully validated by verifying specificity, linearity, LLOQ, intra- and inter-assay precision and accuracy, matrix effect, recovery and stability. Calibration curves with good linearity (r  = 0.9999) over the concentration range from 10 to 5000 ng/ml, intra-assay precision <10% and inter-assay precision <15%, accuracy between 93.0 and 111.6% were obtained with multiple reaction monitoring mode for melamine and cyanuric acid in human urine. The methods were successfully applied to the analysis of urine samples collected from 86 infants and 110 adults.
Keywords: Melamine; Cyanuric acid; Liquid chromatography tandem mass spectrometry; Human urine;

Replacement of acetonitrile by ethanol as solvent in reversed phase chromatography of biomolecules by F. Brettschneider; V. Jankowski; T. Günthner; S. Salem; M. Nierhaus; A. Schulz; W. Zidek; J. Jankowski (763-768).
Acetonitrile, which is a by-product of acrylonitrile synthesis, is the commonly used solvent in ion-pair reversed phase chromatography. In consequence of the decreasing demand for acrylonitrile due to the financial crisis, a worldwide shortage of acetonitrile is observed. Therefore, the aim of this study was to establish ion-pair reversed phase chromatographic assays using alternative eluents for acetonitrile and to decrease costs incurred hereby. We compared the performance of ion-pair reversed phase chromatography using acetonitrile with the alternative eluents methanol, ethanol and n-propanol, using monolithic reversed phase C5 as well as C18 chromatography columns. We used triethylammonium acetate (TEAA) and tetrabutylammonium sulfate (TBA) as representative cationic ion-pair reagents and trifluoroacetic acid (TFA) as representative anionic ion-pair reagent. For covering a large field of applications, we fractionated representative low, middle and high-molecular weight biomolecules, in particular dinucleoside polyphosphates, peptides, proteins and tryptic digested human serum albumin. Whereas the chromatographic characteristics of both methanol and n-propanol were partly insufficient, ethanol was characterised equally or partly even better in the matter of elution strength and separation quality compared to the eluent water–acetonitrile. In conclusion, ethanol is an appropriate alternative for acetonitrile in ion-pair reversed phase chromatography of biomolecules.
Keywords: Dinucleoside polyphosphates; Peptides and proteins; Acetonitrile; Ethanol; Methanol; n-Propanol; Reversed phase chromatography;

Development and validation of a dried blood spot—LC–APCI–MS assay for estimation of canrenone in paediatric samples by Maysa Faisal Suyagh; Prashant Laxman Kole; Jeff Millership; Paul Collier; Henry Halliday; James C. McElnay (769-776).
A selective and sensitive liquid chromatography (LC)–atmospheric pressure chemical ionisation (APCI)–mass spectroscopic (MS) assay of canrenone has been developed and validated employing Dried Blood Spots (DBS) as the sample collection medium. DBS samples were prepared by applying 30 μl of spiked whole blood onto Guthrie cards. A 6 mm disc was punched from the each DBS and extracted with 2 ml of methanolic solution of 17α-methyltestosterone (Internal Standard). The methanolic extract was evaporated to dryness and reconstituted in acetonitrile:water (1:9, v/v). The reconstituted solution was further subjected to solid phase extraction using HLB cartridges. Chromatographic separation was achieved using Waters Sunfire C18 reversed-phase column using isocratic elution, followed by a high organic wash to clear late eluting/highly retained components. The mobile phase consisted of methanol:water (60:40, v/v) pumped at a flow rate of 0.3 ml/min. LC–APCI–MS detection was performed in the selected-ion monitoring (SIM) mode using target ions at m/z 341.1 and 303.3 for canrenone and internal standard respectively. The selectivity of the method was established by analysing DBS samples from 6 different sources (individuals). The calibration curve for canrenone was found to be linear over 25–1000 ng/ml (r  > 0.994). Accuracy (% RE) and precision (% CV) values for within and between day were <20% at the lower limit of quantification (LLQC) and <15% at all other concentrations tested. The LLOQ of the method was validated at 25 ng/ml. Clinical validation of the method was achieved by employing the validated method for analysis of 160 DBS samples from 37 neonatal and paediatric patients.
Keywords: Dried blood spots; Guthrie card; Canrenone; APCI; Clinical validation; Solid phase extraction;

Identification and characterization of oxidation and deamidation sites in monoclonal rat/mouse hybrid antibodies by Vera Timm; Patrick Gruber; Michael Wasiliu; Horst Lindhofer; Dirk Chelius (777-784).
Oxidation of methionine residues and deamidation of asparagine residues are the major causes of chemical degradation of biological pharmaceuticals. The mechanism of these non-enzymatic chemical reactions has been studied in great detail. However, the identification and quantification of oxidation and deamidation sites in a given protein still remains a challenge. In this study, we identified and characterized several oxidation and deamidation sites in a rat/mouse hybrid antibody. We evaluated the effects of the sample preparation on oxidation and deamidation levels and optimized the peptide mapping method to minimize oxidation and deamidation artifacts. Out of a total number of 18 methionine residues, we identified six methionine residues most susceptible to oxidation. We determined the oxidation rate of the six methionine residues using 0.05% H2O2 at different temperatures. Methionine residue 256 of the mouse heavy chain showed the fastest rate of oxidation under those conditions with a half life of approximately 200 min at 4 °C and 27 min at 37 °C. We identified five asparagine residues prone to deamidation under accelerated conditions of pH 8.6 at 37 °C. Kinetic characterization of the deamidation sites showed that asparagine residue 218 of the rat heavy chain exhibited the fastest rate of deamidation with a half live of 1.5 days at pH 8.6 and 37 °C. Analysis of antibody isoforms using free flow electrophoresis showed that deamidation is the major cause of the charged variants of this rat/mouse hybrid antibody.
Keywords: Oxidation; Deamidation; Rat; Mouse; Antibody; Mass spectrometry; Pharmaceutics; Peptide mapping;

Determination of GDC-0449, a small-molecule inhibitor of the Hedgehog signaling pathway, in human plasma by solid phase extraction-liquid chromatographic-tandem mass spectrometry by X. Ding; B. Chou; R.A. Graham; S. Cheeti; S. Percey; L.C. Matassa; S.A. Reuschel; M. Meng; S. Liu; T. Voelker; B.L. Lum; P.J. Rudewicz; C.E.C.A. Hop (785-790).
To support clinical development, a solid phase extraction (SPE) liquid chromatographic-tandem mass spectrometry (LC-MS/MS) method for the determination of GDC-0449 concentrations in human plasma has been developed and validated. Samples (200 μl) were extracted using an Oasis MCX 10 mg 96-well SPE plate and the resulting extracts were analyzed using reverse-phase chromatography coupled with a turbo-ionspray interface. The method was validated over calibration curve range 5–5000 ng/mL. Quadratic regression and 1/x 2 weighing were used. Within-run relative standard deviation (%RSD) was within 10.1% and accuracy ranged from 88.6% to 109.0% of nominal. Between-run %RSD was within 8.6% and accuracy ranged from 92.4% to 105.3% of nominal. Extraction recovery of GDC-0449 was between 88.3% and 91.2% as assessed using quality control sample concentrations. GDC-0449 was stable in plasma for 315 days when stored at −70 °C and stable in reconstituted sample extracts for 117 h when stored at room temperature. Quantitative matrix effect/ion suppression experiment was performed and no significant matrix ion suppression was observed. This assay allows for the determination of GDC-0449 plasma concentrations over a sufficient time period to determine pharmacokinetic parameters at relevant clinical doses.
Keywords: GDC-0449; Hedgehog pathway inhibitor; HPI; LC-MS/MS; 96-Well SPE; Human plasma;

Quantitative determination of helicid in rat biosamples by liquid chromatography electrospray ionization mass spectrometry by Yuanwei Jia; Haitang Xie; Guangji Wang; Jianguo Sun; Wei Wang; Huang Qing; Xuan Wang; Hao Yang; Meijuan Xu; Yi Gu; Chen Yao; Jie Shen (791-797).
A simple liquid chromatography electrospray ionization mass spectrometry (LC–ESI–MS) method with highly improved sensitivities for the determination of helicid in rat bile, urine, feces and most tissues was developed. The tissues and feces were firstly homogenized mechanically using deionized water as the media. Bile, urine, tissues and feces homogenates were extracted by liquid–liquid extraction with n-butyl alcohol for sample preparation. The subsequent analysis procedures were performed on a Shimadzu LCMS2010A system (electrospray ionization single quadrupole mass analyzer). A Luna C18 column (150 mm × 2.00 mm, 5 μm) was used as the analytical column, while a mixture of acetonitrile and ammonium chloride water solution was used as the mobile phase. The proportions of mobile phase were changed timely according to gradient programs. Chlorinated adducts of molecular ions [M+Cl] at m/z 319.00 and 363.05 were used to quantify helicid and bergeninum (internal standard), respectively. The method was validated to be accurate, precise and rugged with good linearity. The proposed method was successfully applied to the preclinical tissue distribution and excretion studies of helicid in rats.
Keywords: Helicid; LC–ESI–MS; Rat biosamples; Tissue distribution; Excretion studies;

Using precipitation by polyamines as an alternative to chromatographic separation in antibody purification processes by Junfen Ma; Hai Hoang; Thomas Myint; Thanmaya Peram; Robert Fahrner; Judy H. Chou (798-806).
Polyamine precipitation conditions for removing host cell protein impurities from the cell culture fluid containing monoclonal antibody were studied. We examined the impact of polyamine concentration, size, structure, cell culture fluid pH and ionic strength. A 96-well microtiter plate based high throughput screening method was developed and used for evaluating different polyamines. Polyallylamine, polyvinylamine, branched polyethyleneimine and poly(dimethylamine-co-epichlorohydrin-ethylenediamine) were identified as efficient precipitants in removing host cell protein impurities. Leveraging from the screening results, we incorporated a polyamine precipitation step into a monoclonal antibody purification process to replace the Protein A chromatography step. The optimization of the overall purification process was performed by taking the mechanisms of both precipitation and chromatographic separation into account. The precipitation-containing process removed a similar amount of process-related impurities, including host cell proteins, DNA, insulin and gentamicin and maintained similar product quality in respect of size and charge variants to chromatography based purification. Overall recovery yield was comparable to the typical Protein A affinity chromatography based antibody purification process.
Keywords: Precipitation; Polyamine; Monoclonal antibody; Purification; Host cell proteins; High throughput screening;

A rapid, sensitive and selective method for the simultaneous quantification of carteolol and dorzolamide in rabbit aqueous humor (AH) and ciliary body (CB) has been developed and validated using reversed phase-high performance liquid chromatography (RP-HPLC) with isocratic elution coupled with atmospheric pressure chemical ionization mass spectrometry/mass spectrometry (APCI-MS/MS). The analytes and nadolol (used as internal standard, IS) were purified from AH by protein precipitation. The sample preparation from CB was based on a two steps extraction procedure at different pH, utilizing a liquid–liquid extraction with a mixture of ethyl acetate, toluene and isopropanol 50:40:10 (v/v) at pH 8, followed by a second extraction with ethyl acetate at pH 11. The combined organic extracts were then back extracted into 0.1% aqueous trifluoroacetic acid (TFA). The accuracy and precision values, calculated from three different sets of quality control samples analyzed in sestuplicate on three different days, were within the generally accepted criteria for analytical methods (<15%). The assay proved to be accurate and precise when applied to the in vivo study of carteolol and dorzolamide in rabbit AH and CB after single administration of an eye drops containing both drugs.
Keywords: Carteolol; Dorzolamide; RP-HPLC/APCI-MS/MS; Quantitative determination; Rabbit aqueous humor; Rabbit ciliary body;

Gas chromatography–ion trap mass spectrometry method for the simultaneous measurement of MDMA (ecstasy) and its metabolites, MDA, HMA, and HMMA in plasma and urine by Daniel Gomes; Paula Guedes de Pinho; Helena Pontes; Luísa Ferreira; Paula Branco; Fernando Remião; Félix Carvalho; M. Lurdes Bastos; Helena Carmo (815-822).
The investigation of 3,4-methylenedioxymethamphetamine (MDMA; ecstasy) abuse requires very robust methods with high sensitivity and wide linearity ranges for the quantification of this drug of abuse and its main metabolites in body fluids. An optimized gas chromatography–ion trap mass spectrometry (GC–IT/MS) methodology with electron impact ionization addressing these issues is presented. The sample preparation involves an enzymatic hydrolysis of urine and plasma for conjugate cleavage, a SPE extraction, and a derivatization process. The method was fully validated in rat plasma and urine. Linearity for a wide concentration range was achieved for MDMA, and the metabolites 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxyamphetamine (HMA) and 4-hydroxy-3-methoxymethamphetamine (HMMA). Limits of quantification were 2 ng/mL in plasma and 3.5 ng/mL in urine using a Selected Ion Monitoring detection mode. Selectivity, accuracy, precision, and recovery met the required criteria for the method validation. This GC–IT/MS method provides high sensitivity and adequate performance characteristics for the simultaneous quantification of MDMA, MDA, HMA and HMMA in the studied matrices.
Keywords: GC–IT/MS; MDMA (ecstasy); Metabolites; Biological fluids;

Simultaneous determination of etoposide and a piperine analogue (PA-1) by UPLC–qTOF-MS: Evidence that PA-1 enhances the oral bioavailability of etoposide in mice by B.S. Sachin; I.A. Najar; S.C. Sharma; M.K. Verma; M.V. Reddy; R. Anand; R.K. Khajuria; S. Koul; R.K. Johri (823-830).
In the present investigation, a UPLC–qTOF-MS/MS method has been developed for the simultaneous determination of etoposide and a piperine analogue, namely, 4-ethyl 5-(3,4-methylenedioxyphenyl)-2E,4E-pentadienoic acid piperidide (PA-1). The analytes were separated on a reverse phase C18 column using methanol–water (72:28, v/v) mobile phase with a flow rate of 250 μL/min. The qTOF-MS was operated under multiple reaction monitoring mode using electro-spray ionization (ESI) technique with positive ion polarity. The major product ions for etoposide and PA-1 were at m/z 185.1350 and 164.1581, respectively. The recovery of the analytes from mouse plasma was optimized using solid phase extraction technique. The total run time was 6 min and the elution of etoposide and PA-1 occurred at 1.24 and 2.84 min, respectively. The calibration curves of etoposide as well as PA-1 were linear over the concentration range of 2–1000 ng/mL (r 2, 0.9829), and 1–1000 ng/mL (r 2, 0.9989), respectively. For etoposide intra-assay and inter-assay accuracy in terms of % bias was in between −7.65 to +6.26, and −7.83 to +5.99, respectively. For PA-1 intra-assay and inter-assay accuracy in terms of % bias was in between −7.01 to +9.10, and −7.36 to +6.71, respectively. The lower limit of quantitation for etoposide and PA-1 were 2.0 and 1.0 ng/mL, respectively. Analytes were stable under various conditions (in autosampler, during freeze–thaw, at room temperature, and under deep-freeze conditions). The method was used for a pharmacokinetic study which showed that PA-1 enhanced the oral bioavailability of etoposide in mice by 2.32-fold.
Keywords: UPLC–qTOF-MS/MS; Etoposide; Piperine analogue; Pharmacokinetics; Bioavailability;

A simple and reproducible high performance liquid chromatography–tandem mass spectrometric method was developed for methocarbamol analysis in human plasma. Methocarbamol and the internal standard (IS) were extracted by a protein precipitation method. Under isocratic separation condition the chromatographic run time was 3.0 min. The calibration curve was linear over a range of 150–12,000 ng/mL with good intraday assay and interday assay precision (CV% < 10.9%). The method was proven to be sensitive and selective for the analysis of methocarbamol in human plasma for bioequivalence study.
Keywords: Methocarbamol; LC/MS/MS; Bioequivalence; Human plasma;

A protease with broad substrate specificity usually produces a complex peptide mixture. However, even-numbered peptides were obtained at high proportion upon papain hydrolysis of fibroin composed of highly repetitive Ala- and Gly-rich blocks. MALDI-TOF and ESI mass spectrometric analysis revealed that the even-numbered peptides were in the forms of di-, tetra-, hexa-, and octa-peptides with repeating units in combination of Ala–Gly, Ser–Gly, Tyr–Gly, and Val–Gly. Application of tandem mass spectrometry identified the sequences of the tetra-peptides to be in the order of Ala–Gly–X–Gly (X = Tyr or Val). Therefore, the substrate specificity of papain and the unique repetitive sequence of fibroin generated the hydrolysate composed of even number of amino acids at a high percentage. In this work, fibroin hydrolysate was investigated as an example of an end product of protein hydrolysis, which provides a clue to understand the fate of peptides in a protein hydrolysate.
Keywords: Fibroin; Hydrolysate; Peptide composition;

Quantification of nortriptyline in plasma by HPLC and fluorescence detection by Patricia Almudever; José-Esteban Peris; Teresa Garrigues; Octavio Diez; Ana Melero; Manuel Alós (841-844).
A simple, sensitive and specific high-performance liquid chromatography method has been developed for the determination of nortriptyline (NT) in plasma samples. The assay involved derivatization with 9H-fluoren-9-ylmethyl chloroformate (Fmoc-Cl) and isocratic reversed-phase (C18) chromatography with fluorescence detection. The developed method required only 100 μl of plasma sample, deproteinized and derivatized in one step. Calibration curves were lineal over the concentration range of 5–5000 ng/ml. The derivatization reaction was performed at room temperature in 20 min and the obtained NT derivative was stable for at least 48 h at room temperature. The within-day and between-day relative standard deviation was below 8%. The limit of detection (LOD) was 2 ng/ml, and the lower limit of quantification (LLOQ) was established at 10 ng/ml. The method was applied on plasma collected from rats, at different time intervals, after intravenous administration of 0.5 mg of NT.
Keywords: Derivatization; Fmoc chloride; Nortriptyline; HPLC;