Development and validation of sensitive LC-MS/MS assays for quantification of HP-#x03B2;-CD in human plasma and CSF
2014; Elsevier BV; Volume: 55; Issue: 7 Linguagem: Inglês
10.1194/jlr.d050278
ISSN1539-7262
AutoresHui Jiang, Rohini Sidhu, Hideji Fujiwara, Marc De Meulder, Ronald P. de Vries, Yong Gong, Mark Kao, Forbes D. Porter, Nicole M. Yanjanin, Nuria Carillo-Carasco, Xin Xu, Elizabeth A. Ottinger, Myra Woolery, Daniel S. Ory, Xuntian Jiang,
Tópico(s)Glycogen Storage Diseases and Myoclonus
Resumo2-Hydroxypropyl-#x03B2;-cyclodextrin (HP-#x03B2;-CD), a widely used excipient for drug formulation, has emerged as an investigational new drug for the treatment of Niemann-Pick type C1 (NPC1) disease, a neurodegenerative cholesterol storage disorder. Development of a sensitive quantitative LC-MS/MS assay to monitor the pharmacokinetics (PKs) of HP-#x03B2;-CD required for clinical trials has been challenging owing to the dispersity of the HP-#x03B2;-CD. To support a phase 1 clinical trial for ICV delivery of HP-#x03B2;-CD in NPC1 patients, novel methods for quantification of HP-#x03B2;-CD in human plasma and cerebrospinal fluid (CSF) using LC-MS/MS were developed and validated: a 2D-LC-in-source fragmentation-MS/MS (2D-LC-IF-MS/MS) assay and a reversed phase ultra performance LC-MS/MS (RP-UPLC-MS/MS) assay. In both assays, protein precipitation and "dilute and shoot" procedures were used to process plasma and CSF, respectively. The assays were fully validated and in close agreement, and allowed determination of PK parameters for HP-#x03B2;-CD. The LC-MS/MS methods are ∼100-fold more sensitive than the current HPLC assay, and were successfully employed to analyze HP-#x03B2;-CD in human plasma and CSF samples to support the phase 1 clinical trial of HP-#x03B2;-CD in NPC1 patients. 2-Hydroxypropyl-#x03B2;-cyclodextrin (HP-#x03B2;-CD), a widely used excipient for drug formulation, has emerged as an investigational new drug for the treatment of Niemann-Pick type C1 (NPC1) disease, a neurodegenerative cholesterol storage disorder. Development of a sensitive quantitative LC-MS/MS assay to monitor the pharmacokinetics (PKs) of HP-#x03B2;-CD required for clinical trials has been challenging owing to the dispersity of the HP-#x03B2;-CD. To support a phase 1 clinical trial for ICV delivery of HP-#x03B2;-CD in NPC1 patients, novel methods for quantification of HP-#x03B2;-CD in human plasma and cerebrospinal fluid (CSF) using LC-MS/MS were developed and validated: a 2D-LC-in-source fragmentation-MS/MS (2D-LC-IF-MS/MS) assay and a reversed phase ultra performance LC-MS/MS (RP-UPLC-MS/MS) assay. In both assays, protein precipitation and "dilute and shoot" procedures were used to process plasma and CSF, respectively. The assays were fully validated and in close agreement, and allowed determination of PK parameters for HP-#x03B2;-CD. The LC-MS/MS methods are ∼100-fold more sensitive than the current HPLC assay, and were successfully employed to analyze HP-#x03B2;-CD in human plasma and CSF samples to support the phase 1 clinical trial of HP-#x03B2;-CD in NPC1 patients. Cyclodextrins are cyclic oligosaccharides consisting of a varying number of α-1-4-linked glucose units. These glucose chains create a cone-like cavity into which compounds may enter and form a water-soluble complex, thus altering the drug's physicochemical properties. The 2-hydroxypropyl-#x03B2;-cyclodextrin (HP-#x03B2;-CD), a hydroxyalkyl derivative of #x03B2;-cyclodextrin, (Fig. 1A) has been widely utilized as an excipient to improve the solubility of poorly water-soluble drugs and to enhance physicochemical properties and chemical stability of drugs, because of its cavity size and greater hydrophilicity. HP-#x03B2;-CD has also emerged as a promising experimental therapy for Niemann-Pick type C1 (NPC1) disease, a rare inherited neurodegenerative disorder with an estimated incidence in Western European and US populations in the order of one in 100,000 live births (1Vanier M.T. Niemann-Pick disease type C.Orphanet J. Rare Dis. 2010; 5: 16Crossref PubMed Scopus (786) Google Scholar). NPC1 disease is characterized by an accumulation of cholesterol and other lipids in the endosomal/lysosomal system, resulting in hepatosplenomegaly, progressive neurologic dysfunction, and early death (2Vanier M.T. Millat G. Niemann-Pick disease type C.Clin. Genet. 2003; 64: 269-281Crossref PubMed Scopus (489) Google Scholar). Treatment with HP-#x03B2;-CD ameliorates cholesterol storage, significantly reducing neurodegeneration and increasing lifespan in murine and feline models of NPC1 disease (3Liu B. Turley S.D. Burns D.K. Miller A.M. Repa J.J. Dietschy J.M. Reversal of defective lysosomal transport in NPC disease ameliorates liver dysfunction and neurodegeneration in the npc1-/- mouse.Proc. Natl. Acad. Sci. USA. 2009; 106: 2377-2382Crossref PubMed Scopus (314) Google Scholar, 4Davidson C.D. Ali N.F. Micsenyi M.C. Stephney G. Renault S. Dobrenis K. Ory D.S. Vanier M.T. Walkley S.U. Chronic cyclodextrin treatment of murine Niemann-Pick C disease ameliorates neuronal cholesterol and glycosphingolipid storage and disease progression.PLoS ONE. 2009; 4: e6951Crossref PubMed Scopus (354) Google Scholar, 5Liu B. Ramirez C.M. Miller A.M. Repa J.J. Turley S.D. Dietschy J.M. Cyclodextrin overcomes the transport defect in nearly every organ of NPC1 mice leading to excretion of sequestered cholesterol as bile acid.J. Lipid Res. 2010; 51: 933-944Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 6Ramirez C.M. Liu B. Taylor A.M. Repa J.J. Burns D.K. Weinberg A.G. Turley S.D. Dietschy J.M. Weekly cyclodextrin administration normalizes cholesterol metabolism in nearly every organ of the Niemann-Pick type C1 mouse and markedly prolongs life.Pediatr. Res. 2010; 68: 309-315Crossref PubMed Scopus (130) Google Scholar, 7Ward S. O'Donnell P. Fernandez S. Vite C.H. 2-hydroxypropyl-beta-cyclodextrin raises hearing threshold in normal cats and in cats with Niemann-Pick type C disease.Pediatr. Res. 2010; 68: 52-56Crossref PubMed Scopus (92) Google Scholar). Based on these studies, individual use investigational new drug applications for HP-#x03B2;-CD have been allowed for eight pediatric patients in the US, and a phase 1 trial for delivery of HP-#x03B2;-CD directly into the lateral ventricle of NPC1 patients was initiated in January 2013 at the National Institutes of Health (NIH) (8Ottinger E.A. Kao M.L. Carrillo-Carrasco N. Yanjanin N. Shankar R.K. Janssen M. Brewster M. Scott I. Xu X. Cradock J. et al.Collaborative development of 2-hydroxypropyl-beta-cyclodextrin for the treatment of Niemann-Pick type C1 disease.Curr. Top. Med. Chem. 2014; 14: 330-339Crossref PubMed Scopus (95) Google Scholar). To support the clinical trial, a reliable assay capable of quantifying HP-#x03B2;-CD in human plasma and cerebrospinal fluid (CSF) was essential. The only bioanalytical assay that has been reported for HP-#x03B2;-CD quantification in human samples involves HPLC using inclusion complex formation and fluorescence detection. A limitation of this assay is the minimum requirement for 1 ml of plasma or urine to reach a lower limit of quantification (LLOQ) of 0.5 #x03BC;g/ml (9Szathmary S.C. Determination of hydroxypropyl-beta-cyclodextrin in plasma and urine by size-exclusion chromatography with post-column complexation.J. Chromatogr. 1989; 487: 99-105Crossref PubMed Scopus (22) Google Scholar). To address the need for a sensitive assay to determine the pharmacokinetics (PKs) of HP-#x03B2;-CD in the context of a clinical trial, we initially developed and validated a novel method utilizing 2D-LC-in-source fragmentation-MS/MS (2D-LC-IF-MS/MS) for sensitivity enhancement with LLOQ for HP-#x03B2;-CD of 10 ng/ml in human plasma and 100 ng/ml for HP-#x03B2;-CD in human CSF, respectively. A more streamlined alternative approach was subsequently developed and validated according to generally accepted regulatory guidances (10.US Department of Health and Human Services Food and Drug Administration. 2001. Guidance for Industry: Bioanalytical Method Validations. Center for Drug Evaluation and Research, Center for Veterinary Medicine. Accessed June 5, 2014, at http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM368107.pdf.Google Scholar, 11Viswanathan C.T. Bansal S. Booth B. DeStefano A.J. Rose M.J. Sailstad J. Shah V.P. Skelly J.P. Swann P.G. Weiner R. Quantitative bioanalytical methods validation and implementation: best practices for chromatographic and ligand binding assays.Pharm. Res. 2007; 24: 1962-1973Crossref PubMed Scopus (614) Google Scholar, 12.European Medicines Agency (EMA). 2011. Guideline on Bioanalytical Method Validation. Accessed June 5, 2014, at http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC500109686.pdf.Google Scholar) to support analysis of samples from the phase 1 trial. The latter assay uses a reversed phase ultra performance liquid chromatography (RP-UPLC)-MS/MS method for quantitative determination of HP-#x03B2;-CD in human plasma and CSF with LLOQs at 50.0 ng/ml and 5.00 #x03BC;g/ml, respectively. We found both assays were robust and reliable. While the 2D-LC-IF-MS/MS method was more sensitive and provided a more complete HP-#x03B2;-CD PK profile, as many postdose plasma samples in clinical study were below the LLOQ of the RP-UPLC-MS/MS method, the RP-UPLC-MS/MS method proved easier to implement and afforded more rapid sample analysis. Herein, we report the development and validation of both assays. The HP-#x03B2;-CD was obtained from Janssen Research and Development (Beerse, Belgium). #x03B2;-Cyclodextrin, sodium hydroxide, ammonium formate, and ammonium carbonate were purchased from Sigma-Aldrich (St. Louis, MO). The d6-propylene oxide was purchased from Polymer Source (Dorval, Montreal, Quebec, Canada). Triethylamine, acetone, methanol, acetonitrile, and formic acid were purchased from EMD Chemicals (Gibbstown, NJ). Methanol was also purchased from Merck (Darmstadt, Germany). Acetonitrile was also purchased from Biosolv (Valkenswaard, The Netherlands). Milli-Q ultrapure water was prepared in-house with a Milli-Q integral water purification system (Billerica, MA). Pooled control human plasma (Na-heparin), human CSF, six lots of individual human plasma, and six lots of individual human CSF were purchased from BioChemed Services (Winchester, VA) or Bioreclamation (Westbury, NY). #x03B2;-Cyclodextrin (0.57 g, 0.499 mmol, 1 equiv) was dissolved in 10% NaOH (1.35 ml, 3.37 mmol, 6.75 equiv) in water. The solution was cooled to 4°C, and d6-propylene oxide (0.411 g, 6.42 mmol, 12.9 equiv) was added over 3.5 h with a Harvard syringe pump (Harvard Apparatus, South Natick, MA). After addition of propylene oxide, the reaction mixture was stirred overnight at 4°C and neutralized with formic acid. The crude product was isolated with solid phase extraction on a Sep-Pak Vac12 cc C18-2g cartridge (Waters, Milford, MA) (20 –90#x0025; methanol in water) to yield 0.37 g of HP-d6-#x03B2;-CD, which contained on average seven d6-hydroxypropyl units per cyclodextrin as determined by 1HNMR (Fig. 1A). A suspension of #x03B2;-cyclodextrin hydrate (0.20 g, 0.17 mmol, 1 equiv) in water (1 ml) containing triethylamine (0.14 ml, 1.0 mmol, 6 equiv) and d3-propylene oxide (0.18 ml, 2.4 mmol, 14 equiv) was heated to 50°C for 1 h and then to 70°C for another hour. The reaction mixture was concentrated to residue. The crude product was suspended and washed repeatedly in acetone. White solid (0.26 g) was isolated, which contained on average 5.9 d3-hydroxypropyl units per cyclodextrin as determined by 1HNMR, as well as a salt in the form of 2-hydroxypropyl-d3-triethylammonium bicarbonate (Fig. 1A). The HP-#x03B2;-CD and internal standard (IS) were accurately weighed and dissolved in water to obtain a 1.00 mg/ml stock solution. Two stock solutions for HP-#x03B2;-CD were prepared independently, and the agreement between the solutions was verified. One solution was used for calibration standards and the other was used for quality control (QC) samples. These solutions were stored at −20°C. The IS spiking solutions were prepared by dilution with water. The calibration standards (10.0–1,500 ng/ml and 0.100–15.0 #x03BC;g/ml in the 2D-LC-IF-MS/MS method for the human plasma and CSF assays, respectively; 50.0–50,000 ng/ml and 5.00–5,000 #x03BC;g/ml in the RP-UPLC-MS/MS method for the human plasma and CSF assays, respectively) and LLOQ, low QC (LQC), medium QC (MQC), high QC (HQC), and dilution QC samples (Tables 2, 3) were prepared by serial dilution after HP-#x03B2;-CD stock solution was spiked into blank biological matrix. All the calibration standards and QCs in plasma and CSF were stored in cryogenic vials at −20 or −80°C after aliquoting.TABLE 2Intra- and inter-run precision and accuracy for HP-#x03B2;-CD in human plasma and human CSF samplesMethod2D-LC-IF-MS/MSRP-UPLC-MS/MSAnalytical run numberMatrixHuman plasmaHuman CSFHuman plasmaHuman CSFQC levelLLOQLQCMQCHQCLLOQLQCMQCHQCLLOQLQCMQCHQCLLOQLQCMQCHQCNominal concentration10.0 ng/ml30.0 ng/ml600 ng/ml1,200 ng/ml0.100 μg/ml0.300 #x03BC;g/ml6.00 #x03BC;g/ml12.0 #x03BC;g/ml50.0 ng/ml125 ng/ml1500 ng/ml40,000 ng/ml5.00 #x03BC;g/ml12.8 #x03BC;g/ml160 #x03BC;g/ml4,000 #x03BC;g/ml1Intra-run mean (n = 6)9.0728.25961,1900.09670.2925.8911.747.71191,440391004.8712.41554,230Intra-run SD0.4661.392548.90.003030.006660.10.2251.593.7347.25750.3770.98811223Intra-run percent CV4.84.94.24.13.12.31.71.93.33.13.31.57.78.07.15.3Intra-run percent bias−2.8−5.9−0.7−0.6−3.3−2.7−1.8−2.8−4.6−4.8−4.0−2.3−2.6−3.1−3.15.82Intra-run mean (n = 6)8.7929.257011500.09930.315.8211.855.31311,55039,3005.0412.41503,870Intra-run SD1.092.2119.937.30.004810.007470.2030.3351.297.1934.67620.3260.5042.4119Intra-run percent CV12.47.63.53.34.82.43.52.82.35.52.21.96.54.11.63.1Intra-run percent bias−12.1−2.7−4.9−4.6−0.73.2−3.0−1.710.64.83.3−1.80.8−3.1−6.3−3.33Intra-run mean (n = 6)10.029.25811,1500.09850.3116.0511.854.31251,45037,0004.8812.51574,020Intra-run SD0.7761.7613.631.90.003460.01280.220.2881.954.7227.912400.2890.3545.180.4Intra-run percent CV7.76.02.32.83.54.13.62.53.63.81.93.45.92.83.22.0Intra-run percent bias0.3−2.8−3.2−4.3−1.53.70.8−2.18.60.0−3.3−7.5−2.4−2.3−1.90.5Inter-runInter-run mean (n = 18)9.5128.95821,1600.09820.3045.9211.752.41251,48038,5004.9312.41544,030Inter-run SD0.9381.7721.743.90.003780.01260.1970.2753.787.3662.51,3600.3230.6337.31204Inter-run percent CV9.96.13.73.83.94.13.32.37.25.94.23.56.65.14.75.1Inter-run percent bias−4.9−3.8−2.9−3.1−1.81.4−1.4−2.24.80.0−1.3−3.8−1.4−3.1−3.80.8 Open table in a new tab TABLE 3Precision and accuracy of dilution QC samples (n = 3) for HP-#x03B2;-CD in human plasma and CSF samplesMethod2D-LC-IF-MS/MSRP-UPLC-MS/MSMatrixHuman plasmaHuman CSFHuman plasmaHuman CSFNominal concentration20,000 ng/ml100 #x03BC;g/ml100 #x03BC;g/ml400,000 ng/ml40,000 #x03BC;g/mlDilution factor20202001010Mean (n = 3)21,10094.894396,00034,900SD5690.71.238,810891Percent CV2.70.71.32.22.6Percent bias5.7−5.2−6.0−1.0−12.8 Open table in a new tab For the plasma assay, to 200 #x03BC;l of plasma sample, IS spiking solution (50 #x03BC;l of 5 #x03BC;g/ml HP-d6-#x03B2;-CD in water in the 2D-LC-IF-MS/MS method or 20 #x03BC;l of 2.00 #x03BC;g/ml HP-d3-#x03B2;-CD in water in the RP-UPLC-MS/MS method) was added, except for the blank without IS in which water (50 #x03BC;l in the 2D-LC-IF-MS/MS method and 20 #x03BC;l in the RP-UPLC-MS/MS method) was used, followed by adding methanol (750 and 800 #x03BC;l in the 2D-LC-IF-MS/MS method and the RP-UPLC-MS/MS method, respectively). The mixtures were vortexed and then centrifuged. The supernatants were then transferred to clean tubes and evaporated to dryness under a stream of nitrogen. The residue was reconstituted with 200 #x03BC;l of water. For the CSF assay in the 2D-LC-IF-MS/MS method, 50 l of CSF sample was mixed well with 100 #x03BC;l of IS spiking solution (2.5 #x03BC;g/ml HP-d6-#x03B2;-CD in water), and the blank without IS was mixed with 100 l of water. In the RP-UPLC-MS/MS method, 50 l of CSF sample was diluted with 1,900 #x03BC;l of water, and 100 #x03BC;l of the diluted sample was mixed well with 50 #x03BC;l of IS spiking solution (20.0 #x03BC;g/ml HP-d3-#x03B2;-CD in water), except for the blank without IS in which 50 l of water was used, and to these samples another 100 #x03BC;l water was added. Each accuracy and precision run included eight calibration standards in duplicate for the 2D-LC-IF-MS/MS method, ten calibration standards in single for the RP-UPLC-MS/MS method, and six replicates of QC samples at the concentration levels of LLOQ, LQC, MQC, and HQC. At least one blank and one blank with IS were included in each run. The plasma samples with a concentration above the highest calibrator and dilution QC sample were diluted 10- or 20-fold with blank plasma prior to extraction. The CSF samples with concentrations above the highest calibrator and dilution QC sample were diluted 10-, 20-, or 200-fold with water or blank CSF prior to extraction. A 4000QTRAP and an API 4000 mass spectrometer (AB/MDS-Sciex, Concord, Ontario, Canada) with TurboIonSprayTM interface operated in positive ionization mode were used in the 2D-LC-IF-MS/MS method and the RP-UPLC-MS/MS method, respectively. In the 2D-LC-IF-MS/MS method, the multiple reaction monitoring (MRM) mass transitions m/z 203.0→97.0 and m/z 209.0→97.0 were used for HP-#x03B2;-CD and HP-d6-#x03B2;-CD, respectively, with a dwell time of 100 ms for each mass transition. The following precursor product ion transitions were used in the RP-UPLC-MS/MS method for MRM: HP-#x03B2;-CD, m/z 1326.5→383.0; and HP-d3-#x03B2;-CD, m/z 1335.6→386.0; with a dwell time of 200 ms for each mass transition of the analyte and IS. The mass spectrometer was operated at unit mass resolution for both the first and third quadrupole. The optimized instrument parameters are listed in Table 1.TABLE 1Optimized mass spectrometric parametersMethodMass SpectrometerCURTEMGS1GS2iheCADISDPEPCECXP2D-LC-IF-MS/MS4000QTRAP20500°C3520ONMedium5,00025491615RP-UPLC-MS/MSAPI 400030450°C4050ON65,00081104512CAD, collision gas; CE, collision energy; CUR, curtain gas; CXP, collision cell exit potential; DP, declustering potential; EP, entrance potential; GS1, ion source gas 1 (nebulizer gas); GS2, ion source gas 2 (auxiliary gas); ihe, interface heater; IS IonSpray voltage; TEM, temperature. Open table in a new tab CAD, collision gas; CE, collision energy; CUR, curtain gas; CXP, collision cell exit potential; DP, declustering potential; EP, entrance potential; GS1, ion source gas 1 (nebulizer gas); GS2, ion source gas 2 (auxiliary gas); ihe, interface heater; IS IonSpray voltage; TEM, temperature. A Shimadzu Prominence HPLC system with a CBM-20A system controller, 4 LC-20AD pumps, a SIL-20ACHT autosampler, a rack changer (Shimadzu Scientific Instruments, Columbia, MD), and two six-port valves (Valco Instruments, Houston, TX) were used to separate HP-#x03B2;-CD and HP-d6-#x03B2;-CD from the biological matrix. The chromatography was performed at ambient temperature using a C18 guard column (4 × 3.0 mm, Phenomenex, Torrance, CA) and Atlantis hydrophilic interaction liquid chromatography (HILIC) silica column (3 × 50 mm, 3 #x03BC;m; Waters) as first and second dimensions, respectively. The compartment of the autosampler was set at 4°C. Figure 2 is a schematic of the column and switching valve arrangement for 2D-LC. For the first dimension, mobile phase A (10 mM ammonium formate and 0.1#x0025; formic acid in water) and mobile phase B [0.1#x0025; formic acid in methanol-acetonitrile (4:1)] were operated with a gradient elution as follows: 0–0.3 min 0#x0025; B, 0.3–2.0 min 0–20#x0025; B, 2.0–2.1 min 20–100#x0025; B, 2.1–6.0 min 100#x0025; B, 6.0–6.1 min 100–0#x0025; B, and 6.1–7.5 min 0#x0025; B at a flow rate of 0.6 ml/min. The solvent gradient for second dimension LC using 10 mM ammonium formate and 0.1#x0025; formic acid in methanol-water (4:1) (phase C) and acetonitrile (phase D) at a flow rate of 0.60 ml/min was as follows: 0–2.1 min 88#x0025; D, 2.1–5.0 min 88–40#x0025; D, 5.0–5.5 min 40#x0025; D, 5.5–5.6 min 40–88#x0025; D, and 5.6–7.5 min 88#x0025; D. The valve 1 switched from position A to B at 1.6 min and back to position A at 1.8 min. The valve 2 switched from position A to B at 5.0 min and back to position A at 6.0 min. The LC system was an Acquity UPLC® (Waters) consisting of binary solvent manager and sample manager. The separations were carried out using an Acquity UPLC BEH Shield RP18 column (2.1 × 100 mm, 1.7 #x03BC;m; Waters) at 40°C. The components were eluted with a 4 min gradient program at a flow rate of 0.50 ml/min with an injection volume of 10 #x03BC;l for plasma and 5 #x03BC;l for CSF. The mobile phase was composed of mobile phase A (10.0 mM ammonium carbonate in water) and mobile phase B (acetonitrile). The linear gradient was as follows: 0–0.5 min 1#x0025; B; 0.5–2.0 min 1–55#x0025; B; 2.0–2.1 min 55–98#x0025; B; 2.1–2.8 min 98#x0025; B; 2.8–2.9 min 98–1#x0025; B, and 2.9–4.0 min 1#x0025; B. This clinical study was approved by the Institutional Review Board of the Eunice Kennedy Shriver National Institute of Child Health and Human Development. Permission from guardians and assent, when possible, were obtained from all participants. The study was posted on ClinicalTrials.gov (NCT01747135) and use of HP-#x03B2;-CD was covered under investigational new drug (IND) 113273. Three NPC1 subjects were admitted to the NIH Clinical Center Intensive Care Unit and an Ommaya reservoir was surgically placed on the nondominant side. The 50 mg HP-#x03B2;-CD dose was prepared in 5 ml of an isotonic salt solution. Vehicle (saline) and HP-#x03B2;-CD doses were administered ICV via the Ommaya reservoir. CSF and blood, 1.5 and 2 ml respectively, were collected at 0.25, 0.5, 1, 3, 8, 24, 36, and 48 h predose, and 72 h postdose. PK parameters of HP-#x03B2;-CD for CSF and plasma were calculated with the noncompartmental approach using Phoenix WinNonlin software (version 6.2) (Certara, St. Louis, MO). Briefly, the area under the concentration-time curve (AUC) was calculated using the linear trapezoidal method. Where warranted, the slope of the apparent terminal phase was estimated by log-linear regression using at least three data points and the terminal rate constant (λ) was derived from the slope. AUC0-∞ was estimated as the sum of the AUC0-t (where t is the time of the last measurable concentration) and concentration after time t (Ct)/λ. The apparent terminal half-life (t1/2) was calculated as 0.693/λ. For the CSF data, the clearance (CL) of HP-#x03B2;-CD was calculated as dose/AUC0-∞, and the volume of distribution at steady-state (Vdss) was calculated as (AUMC0-∞ /AUC0-∞) × CL, where AUMC0-∞ is the area under the first moment curve from zero to infinity. For the plasma data, the systemic exposure of HP-#x03B2;-CD was assessed by the maximum drug concentration (Cmax), time to reach Cmax (Tmax), AUC, and t1/2. HP-#x03B2;-CD is a heterogeneous complex mixture of homologs and isomers, in which there are a variable number of 2-hydroxypropyl groups substituted at different positions of the sugar moieties. Previously, unambiguous, sensitive, and quantitative detection of the polysaccharide mixture as a whole was not feasible due to the dispersity of the signals that are distributed over a large number of homologs and isomers. In theory, there are 2,097,151 possible homologs and isomers in the HP-#x03B2;-CD mixture based on combination of variance in degree of substitution and the possible sites of the substitution (13Szente L. Szejtli J. Highly soluble cyclodextrin derivatives: chemistry, properties, and trends in development.Adv. Drug Deliv. Rev. 1999; 36: 17-28Crossref PubMed Scopus (463) Google Scholar). In order to quantify total HP-#x03B2;-CD concentrations with LC-MS/MS, innovative approaches needed to be developed and investigated. One approach was in-source fragmentation of all the HP-#x03B2;-CD homologs and isomers present into common building blocks. Alternatively, it was investigated to determine whether one single homolog could be used as a probe compound for the quantification of the total of all the HP-#x03B2;-CD homologs. Mobile phase additives have a strong influence on HP-#x03B2;-CD distribution profiles in their ESI mass spectra. Due to relatively weak proton affinity, even in the presence of 0.1#x0025; formic acid, HP-#x03B2;-CD formed abundant sodium adducts and a low abundance of protonated molecular ions that were most often used for mass spectrometric detection in positive ion mode. In the mobile phase of second dimensional LC of 2D-LC-IF-MS/MS (10 mM of ammonium formate and 0.1#x0025; of formic acid as additives in mobile C), ammonium-, proton-, and sodium-adduct ions were observed; the protonated ions were probably generated from neutral loss of ammonia from ammonium adduct ions. In the mobile phase of the RP-UPLC-MS/MS method (10.0 mM ammonium carbonate as additive in mobile phase A), abundant ammonium adducts were formed. The number of 2-hydroxypropyl groups per #x03B2;-cyclodextrin unit ranges from three to nine in the mobile phases of the second dimension LC of 2D-LC-IF-MS/MS (Fig. 1B), and one to seven in the mobile phase of the RP-UPLC-MS/MS method (Fig. 1C). Although the HP-#x03B2;-CD with the same substitution degree appears as single peak in mass spectrum, it is a complex mixture composed of a large number of isobaric isomers. The efficiency of adduct formation for these isomers is different, leading to a different substitution pattern observed in the mass spectra of HP-#x03B2;-CD in different solutions. Sodium adducts were easily formed in the ion source; however, they were not suitable for MS/MS detection due to their poor fragmentation. For our methods, ammonium- and proton-adduct ions were employed as precursor ions for MS/MS detection. When the voltage is significantly increased in the high-pressure region between the sample cone and skimmer in an atmospheric pressure ionization source, the ions passing through this region may dissociate when they collide with solvent or air molecules. This technique is called in-source fragmentation- or in-source collision-induced dissociation. In-source fragmentation has been used in structural elucidation (14Hsu F.F. Turk J. Stewart M.E. Downing D.T. Structural studies on ceramides as lithiated adducts by low energy collisional-activated dissociation tandem mass spectrometry with electrospray ionization.J. Am. Soc. Mass Spectrom. 2002; 13: 680-695Crossref PubMed Scopus (79) Google Scholar), compound identification (15Farwanah H. Wirtz J. Kolter T. Raith K. Neubert R.H. Sandhoff K. Normal phase liquid chromatography coupled to quadrupole time of flight atmospheric pressure chemical ionization mass spectrometry for separation, detection and mass spectrometric profiling of neutral sphingolipids and cholesterol.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2009; 877: 2976-2982Crossref PubMed Scopus (49) Google Scholar, 16Altman E. Li J. Characterization of polysaccharides using mass spectrometry for bacterial serotyping.Methods Mol. Biol. 2010; 600: 245-257Crossref PubMed Scopus (2) Google Scholar, 17Prandi B. Farioli L. Tedeschi T. Pastorello E.A. Sforza S. Simulated gastrointestinal digestion of Pru ar 3 apricot allergen: assessment of allergen resistance and characterization of the peptides by ultra-performance liquid chromatography/electrospray ionisation mass spectrometry.Rapid Commun. Mass Spectrom. 2012; 26: 2905-2912Crossref PubMed Scopus (8) Google Scholar), compound profiling (18Little J.L. Wempe M.F. Buchanan C.M. Liquid chromatography-mass spectrometry/mass spectrometry method development for drug metabolism studies: Examining lipid matrix ionization effects in plasma.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2006; 833: 219-230Crossref PubMed Scopus (208) Google Scholar), and bioanalysis (19Li H. Rose M.J. Holder J.R. Wright M. Miranda L.P. James C.A. Direct quantitative analysis of a 20 kDa PEGylated human calcitonin gene peptide antagonist in cynomolgus monkey serum using in-source CID and UPLC-MS/MS.J. Am. Soc. Mass Spectrom. 2011; 22: 1660-1667Crossref PubMed Scopus (19) Google Scholar). In-source fragmentation of HP-#x03B2;-CD, which was promoted by increasing declustering potentials on a 4000QTRAP mass spectrometer, led to glycosidic-bond cleavage and simultaneous fragmentation of all the HP-#x03B2;-CD ammonium- and proton-adduct ions. The in-source fragmentation effectively generated abundant glucopyranose unit ions (Fig. 1D), and the complex mixture of homologs and isomers was simplified to several building blocks of HP-#x03B2;-CD. The m/z 203 ion was a 2-hydroxypropyl substituted dihydropyrylium ion representing the simplest building block for HP-#x03B2;-CD, and it was chosen as a surrogate for HP-#x03B2;-CD and as the precursor for further collision-activated dissociation. The structure of the major component of the m/z 203 ion was proposed based on its product ion spectrum (Fig. 1E). The IS HP-d6-#x03B2;-CD demonstrated similar in-source fragmentation and product ion spectra of the m/z 209 ion. The major product ion at m/z 97, a 4-hydroxypyrylium, was observed for the m/z 203 and m/z 209 ions. Hence, MRM transitions m/z 203→97 and m/z 209→97 were selected for detection of HP-#x03B2;-CD and the IS, respectively. As an alternative approach in the RP-UPLC-MS/MS method, individual HP-#x03B2;-CD substituted forms, instead of the sum of all substituted forms, were monitored in MRM mode. Method development data revealed that monitoring any of the individual substituted forms was representative for the total of all HP-#x03B2;-CD substituted forms and provided similar results as when using the in-source fragmentation approach or fluorescence detection on condition that the same lot of compound is used for the quantitation as for dosing in the study. Eventually, HP-#x03B2;-CD containing three 2-hydroxypropyl groups was selected as a probe compound in MRM detection, as this isoform, although it is not the most abundant ion in the ESI mass spectrum, provided the best sensitivity in MRM mode. The MRM transitions m/z 1,326.5→383 and m/z 1,335.6→386 were
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