Development and validation of sensitive LC-MS/MS assays for quantification of HP-β-CD in human plasma and CSF

2-Hydroxypropyl-β-cyclodextrin (HP-β-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-β-CD required for clinical trials has been challenging owing to the dispersity of the HP-β-CD. To support a phase 1 clinical trial for ICV delivery of HP-β-CD in NPC1 patients, novel methods for quantification of HP-β-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-β-CD. The LC-MS/MS methods are ∼100-fold more sensitive than the current HPLC assay, and were successfully employed to analyze HP-β-CD in human plasma and CSF samples to support the phase 1 clinical trial of HP-β-CD in NPC1 patients.     

Determination of 2-hydroxypropyl-γ-cyclodextrin in plasma of cynomolgus monkeys after oral administration by gas chromatography–mass spectrometry

This report describes a specific and highly sensitive gas chromatography–mass spectrometry (GC–MS) assay for the analysis of industrially produced 2-hydroxypropyl-γ-cyclodextrin, a heterogeneous mixture of homologues and isomers, in plasma of cynomolgus monkeys. Instead of analyzing the polysaccharide mixture as a whole, in a first step the HP-γ-cyclodextrin mixture, together with the internal standard (2,6-di-O-methyl-β-cyclodextrin), was deuteromethylated, and in a second step hydrolyzed with hydrochloric acid to the respective monosaccharides. The resulting reaction mixture was trimethylsilylated to 1,4-bis(O-trimethylsilyl)-2,3-bis-O-deuteromethyl-6-O-2′-deuteromethoxypropylglucose (representative for HP-γ-CD) and 1,4-bis-(O-trimethylsilyl)-bis-2,6-O-methyl-3-O-deuteromethylglucose (representative for the internal standard), respectively, and analyzed by GC–MS. The limit of quantification of this assay was 20 nmol/l.     

Cerebrospinal Fluid Steroidomics: Are Bioactive Bile Acids Present in Brain?

In this study we have profiled the free sterol content of cerebrospinal fluid by a combination of charge tagging and liquid chromatography-tandem mass spectrometry. Surprisingly, the most abundant cholesterol metabolites were found to be C₂₇ and C₂₄ intermediates of the bile acid biosynthetic pathways with structures corresponding to 7α-hydroxy-3-oxocholest-4-en-26-oic acid (7.170 ± 2.826 ng/ml, mean ± S.D., six subjects), 3β-hydroxycholest-5-en-26-oic acid (0.416 ± 0.193 ng/ml), 7α,x-dihydroxy-3-oxocholest-4-en-26-oic acid (1.330 ± 0.543 ng/ml), and 7α-hydroxy-3-oxochol-4-en-24-oic acid (0.172 ± 0.085 ng/ml), and the C₂₆ sterol 7α-hydroxy-26-norcholest-4-ene-3,x-dione (0.204 ± 0.083 ng/ml), where x is an oxygen atom either on the CD rings or more likely on the C-17 side chain. The ability of intermediates of the bile acid biosynthetic pathways to activate the liver X receptors (LXRs) and the farnesoid X receptor was also evaluated. The acidic cholesterol metabolites 3β-hydroxycholest-5-en-26-oic acid and 3β,7α-dihydroxycholest-5-en-26-oic acid were found to activate LXR in a luciferase assay, but the major metabolite identified in this study, i.e. 7α-hydroxy-3-oxocholest-4-en-26-oic acid, was not an LXR ligand. 7α-Hydroxy-3-oxocholest-4-en-26-oic acid is formed from 3β,7α-dihydroxycholest-5-en-26-oic acid in a reaction catalyzed by 3β-hydroxy-Δ⁵-C₂₇-steroid dehydrogenase (HSD3B7), which may thus represent a deactivation pathway of LXR ligands in brain. Significantly, LXR activation has been found to reduce the symptoms of Alzheimer disease (Fan, J., Donkin, J., and Wellington C. (2009) Biofactors 35, 239–248); thus, cholesterol metabolites may play an important role in the etiology of Alzheimer disease.     

Ditosylate Salt of Itraconazole and Dissolution Enhancement Using Cyclodextrins

Salt formation has been a promising approach for improving the solubility of poorly soluble acidic and basic drugs. The aim of the present study was to prepare the salt form of itraconazole (ITZ), a hydrophobic drug to improve the solubility and hence dissolution performance. Itraconazolium ditolenesulfonate salt (ITZDITOS) was synthesized from ITZ using acid addition reaction with p-toluenesulfonic acid. Salt characterization was performed using ¹H NMR, mass spectrometry, Fourier transform infrared spectroscopy, differential scanning calorimetry, and X-ray diffraction. The particle size and morphology was studied using dynamic light scattering technique and scanning electron microscopy, respectively. The solubility of the salt in water and various pharmaceutical solvents was found multifold than ITZ. The dissolution study exhibited 5.5-fold greater percentage release value in 3 h of ITZDITOS (44.53%) as compared with ITZ (8.54%). Results of in vitro antifungal studies using broth microdilution technique indicate that ITZDITOS possessed similar antifungal profile as that of ITZ when tested against four fungal pathogens. Furthermore, the physical mixtures of ITZDITOS with two cyclodextrins, β-cyclodextrin (β-CD), and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) were prepared in different molar ratios and were evaluated for in vitro release. It was observed that in only 30 min of dissolution study, about 74 and 81% of drug was released from 1:3 molar ratios of ITZDITOS with β-CD and ITZDITOS with HP-β-CD, respectively, which was distinctly higher than the drug released from ITZ commercial capsules (70%). The findings warrant further preclinical and clinical studies on ITZDITOS so that it can be established as an alternative to ITZ for developing oral formulations.KEY WORDS: antifungal, BCS class II, dissolution rate, insoluble, itraconazole     

A modified HPLC technique for simultaneous measurement of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in cerebrospinal fluid, platelet and plasma.

A sensitive, reliable and simplified HPLC assay for simultaneous measurement of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in human cerebrospinal fluid (CSF), platelets and plasma is described. Perchloric acid is used for one step precipitation of proteins and extraction of 5-HT and 5-HIAA. Precision of the assay has been increased by calibration of the instrument using serotonin-free plasma spiked with known amount of standards and N-w-methyl-5-hydroxytryptamine as internal standard. Integration of the peaks and calculations are achieved by a preprogrammed data module using ratio method. As little as 20 pg/ml of serotonin in the deproteinated sample can be detected using this procedure. In a group of surgical patients, plasma 5-HT concentration is (Mean +/- S D) 3.4 +/- 2.7 ng/ml and that of platelet 748.3 +/- 448.3 ng/10(9) platelets. In CSF, 5-HT is found to be 3.3 +/- 3.4 ng/ml and 5-HIAA is 15.1 +/- 7.3 ng/ml. A good correlation (r = 0.648, p less than .0001) is observed between 5-HT and 5-HIAA in CSF.     

Simultaneous determination of nimesulide and hydroxynimesulide in rat plasma,cerebrospinal fluid and brain by liquid chromatography using solid-phase extraction

A liquid chromatographic method with UV detection for the quantification of nimesulide (N) and hydroxynimesulide (M1) in rat plasma, cerebrospinal fluid (CSF) and brain tissue is reported. Plasma samples (250 microl) and brain homogenates added with the right amount of the internal standard (I.S., 2'-(cyclohexyloxy)-4'-nitrophenyl methanesulphonanilide, NS398) are extracted on C(18) disposable cartridges by solid-phase extraction (SPE), while CSF samples are analyzed without any extraction. The separation is performed at room temperature on a Waters Symmetry C(18) 3.5 microm (150x4.6 mm I.D.) column with acetonitrile-sodium citrate buffer pH 3.00 (53:47, v/v) as mobile phase, at a flow-rate of 1.1 ml/min and detection at 240 nm. The retention times are 3.3, 6.0 and 9.9 min for M1, N and I.S., respectively. The lower limits of quantitation for either nimesulide and M1 are 25 ng/ml for plasma, 20 ng/ml for CSF and 25 ng/g for brain tissue. The calibration curves are linear up to 10,000 ng/ml for plasma, 5000 ng/ml for CSF and 5000 ng/g for brain tissue. This new assay can be applied to the study of the role of nimesulide in the modulation of neuroinflammatory processes.     

Quantitation of plasma mevalonic acid using gas chromatography-electron capture mass spectrometry

Circulating concentrations of mevalonic acid (MVA) change in parallel with, and may be used as a marker of cholesterol biosynthesis. Plasma MVA levels have been quantified using a sensitive and specific capillary gas chromatography-electron capture mass spectrometric assay. The detection limit for MVA in plasma is 100 pg/ml; the intra-assay variation is 5.11%; the inter-assay variation is 7.7%. Using this assay, the mean plasma MVA in 15 normolipidemic subjects was 2.37 +/- 1.2 ng/ml (range 0.41-5.31 ng/ml). Administration of 40 mg of simvastatin (an HMG-CoA reductase inhibitor) significantly accenutated the diurnal decrease in plasma MVA levels. This assay may be useful in investigating cholesterol synthesis rates in different dyslipidemias and individual responses of HMG-CoA reductase-inhibiting drugs.     

Stereoselective analysis of hydroxybupropion and application to drug interaction studies

A sensitive and stereoselective assay has been developed for the quantitation of the enantiomers of hydroxybupropion, an active metabolite of bupropion, in human plasma. The assay used liquid-liquid extraction and a Cyclobond I 2000 HPLC column with a mobile phase containing 3% acetonitrile, 0.5% triethylamine, and 20 mM ammonium acetate (pH 3.8). The technique was linear over the concentration range of 12.5-500 ng/ml for (2R,3R)- and (2S,3S)-hydroxybupropion. The method was reproducible as both interday and intraday variabilities were less than 10% for both hydroxybupropion enantiomers. Overall extraction recovery of hydroxybupropion enantiomers and the internal standard phenacetin from plasma was greater than 80% and reproducible over the concentration range of 12.5-500 ng/ml for each enantiomer. The limit of quantification (LOQ) of hydroxybupropion enantiomers was 12.5 ng/ml. The stereoselective pharmacokinetics of both (2R,3R)- and (2S,3S)-hydroxybupropion in healthy male subjects (n = 16) were investigated after a single dose of (rac)-bupropion either alone or during rifampicin administration. (2R,3R)-Hydroxybupropion was the predominant enantiomer present in plasma. A stereoselective effect of rifampicin on hydroxybupropion concentrations was observed, with rifampicin influencing the pharmacokinetics of each hydroxybupropion enantiomer in a different manner. The ratio of (2R,3R)-hydroxybupropion (AUC(0-24)) to (2S,3S)-hydroxybupropion (AUC(0-24)) increased from 4.9 +/- 1.6 to 8.3 +/- 1.9 during rifampicin administration (P < 0.001). A time-dependent change in the hydroxybupropion enantiomeric ratio was observed after (rac)-bupropion administration both before and during rifampicin coadministration, with an increase in the relative proportion of (2S,3S)-hydroxybupropion over the 24 h postdose period.     

Simple and sensitive liquid chromatography-tandem mass spectrometry method for determination of the S(+)- and R(-)-enantiomers of baclofen in human plasma and cerebrospinal fluid

A simple and sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) method to determine the enantiomers of the muscle relaxant baclofen in human plasma and cerebrospinal fluid (CSF) has been developed. A commercially available ultrafiltration membrane is used to prepare the sample. A chiral CROWNPAK CR(+) stationary phase column is then used to perform complete resolution of the S(+)- and R(-)-enantiomers of baclofen. This method was used to analyze human plasma and CSF spiked with baclofen, and the calibration curves for both biologic samples were linear over a concentration range of 0.15-150 ng enantiomer/ml. The lower limit of quantification was 0.15 ng enantiomer/ml in both fluids. Finally, the method was tested with an artificial CSF as an alternative to authentic human CSF. The results showed that no matrix effects and no interfering peaks were observed using this artificial CSF.     

Resolution and quantitation of pentazocine enantiomers in human serum by reversed-phase high-performance liquid chromatography using sulfated β-cyclodextrin as chiral mobile phase additive and solid-phase extraction

A sensitive and stereospecific HPLC method was developed for the analysis of (−)- and (+)-pentazocine in human serum. The assay involves the use of a phenyl solid-phase extraction column for serum sample clean-up prior to HPLC analysis. Chromatographic resolution of the pentazocine enantiomers was performed on a octadecylsilane column with sulfated-β-cyclodextrin (S-β-CD) as the chiral mobile phase additive. The composition of the mobile phase was aqueous 10 mM potassium dihydrogenphosphate buffer pH 5.8 (adjusted with phosphoric acid)–absolute ethanol (80:20, v/v) containing 10 mM S-β-CD at a flow-rate of 0.7 ml/min. Recoveries of (−)- and (+)-pentazocine were in the range of 91–93%. Linear calibration curves were obtained in the 20–400 ng/ml range for each enantiomer in serum. The detection limit based on S/N=3 was 15 ng/ml for each pentazocine enantiomer in serum with UV detection at 220 nm. The limit of quantitation for each enantiomer was 20 ng/ml. Precision calculated as R.S.D. and accuracy calculated as error were in the range 0.9–7.0% and 1.2–6.2%, respectively, for the (−)-enantiomer and 0.8– 7.6% and 1.2–4.6%, respectively, for the (+)-enantiomer (n=3).     

Liquid chromatographic-tandem mass spectrometric assay for the simultaneous determination of didanosine and stavudine in human plasma, bronchoalveolar lavage fluid, alveolar cells, peripheral blood mononuclear cells, seminal plasma, cerebrospinal fluid and tonsil tissue

We have developed a sensitive, high-pressure liquid chromatographic-tandem mass spectrometric (LC/MS/MS) method for the simultaneous determination of didanosine (ddI) and stavudine (d4T) in human plasma, bronchoalveolar lavage fluid (BALF), alveolar cells (AC), peripheral blood mononuclear cells (PBMC), seminal plasma, cerebrospinal fluid (CSF), and tonsil tissue. Plasma, AC, PBMC and CSF were run with an isocratic HPLC method, while BALF supernatant, semen, and tonsil tissue utilized a gradient elution. Samples were prepared by solid phase extraction. Detection was by electrospray positive ionization with multiple reaction monitoring mode. The lower limits of quantitation for both ddI and d4T were 2.0 ng/ml in plasma; 0.5 ng/ml in CSF; 0.4 ng/ml in AC, PBMC, and BALF; 1.0 ng/ml in seminal plasma; and 0.01 ng/mg in tonsil tissue.     

Evaluating Amyloid-β Oligomers in Cerebrospinal Fluid as a Biomarker for Alzheimer’s Disease

The current study evaluated amyloid-β oligomers (Aβo) in cerebrospinal fluid as a clinical biomarker for Alzheimer’s disease (AD). We developed a highly sensitive Aβo ELISA using the same N-terminal monoclonal antibody (82E1) for capture and detection. CSF samples from patients with AD, mild cognitive impairment (MCI), and healthy controls were examined. The assay was specific for oligomerized Aβ with a lower limit of quantification of 200 fg/ml, and the assay signal showed a tight correlation with synthetic Aβo levels. Three clinical materials of well characterized AD patients (n = 199) and cognitively healthy controls (n = 148) from different clinical centers were included, together with a clinical material of patients with MCI (n = 165). Aβo levels were elevated in the all three AD-control comparisons although with a large overlap and a separation from controls that was far from complete. Patients with MCI who later converted to AD had increased Aβo levels on a group level but several samples had undetectable levels. These results indicate that presence of high or measurable Aβo levels in CSF is clearly associated with AD, but the overlap is too large for the test to have any diagnostic potential on its own.     

Simultaneous Determination of Oxysterols,Cholesterol and 25-Hydroxy-Vitamin D3 in Human Plasma by LC-UV-MS

Background

Oxysterols are promising biomarkers of neurodegenerative diseases that are linked with cholesterol and vitamin D metabolism. There is an unmet need for methods capable of sensitive, and simultaneous quantitation of multiple oxysterols, vitamin D and cholesterol pathway biomarkers.

Methods

A method for simultaneous determination of 5 major oxysterols, 25-hydroxy vitamin D3 and cholesterol in human plasma was developed. Total oxysterols were prepared by room temperature saponification followed by solid phase extraction from plasma spiked with deuterated internal standards. Oxysterols were resolved by reverse phase HPLC using a methanol/water/0.1% formic acid gradient. Oxysterols and 25-hydroxy vitamin D3 were detected with atmospheric pressure chemical ionization mass spectrometry in positive ion mode; in-series photodiode array detection at 204nm was used for cholesterol. Method validation studies were performed. Oxysterol levels in 220 plasma samples from healthy control subjects, multiple sclerosis and other neurological disorders patients were quantitated.

Results

Our method quantitated 5 oxysterols, cholesterol and 25-hydroxy vitamin D3 from 200 μL plasma in 35 minutes. Recoveries were >85% for all analytes and internal standards. The limits of detection were 3-10 ng/mL for oxysterols and 25-hydroxy vitamin D3 and 1 μg/mL for simultaneous detection of cholesterol. Analytical imprecision was <10 %CV for 24(S)-, 25-, 27-, 7α-hydroxycholesterol (HC) and cholesterol and ≤15 % for 7-keto-cholesterol. Multiple Sclerosis and other neurological disorder patients had lower 27-hydroxycholesterol levels compared to controls whereas 7α-hydroxycholesterol was lower specifically in Multiple Sclerosis.

Conclusion

The method is suitable for measuring plasma oxysterols levels in human health and disease. Analysis of human plasma indicates that the oxysterol, bile acid precursors 7α-hydroxycholesterol and 27-hydroxycholesterol are lower in Multiple Sclerosis and may serve as potential biomarkers of disease.     

Stereoselective analysis of bupropion and hydroxybupropion in human plasma and urine by LC/MS/MS

A sensitive, stereoselective assay using solid phase extraction and LC-MS-MS was developed and validated for the analysis of (R)- and (S)-bupropion and its major metabolite (R,R)- and (S,S)-hydroxybupropion in human plasma and urine. Plasma or glucuronidase-hydrolyzed urine was acidified, then extracted using a Waters Oasis MCX solid phase 96-well plate. HPLC separation used an alpha(1)-acid glycoprotein column, a gradient mobile phase of methanol and aqueous ammonium formate, and analytes were detected by electrospray ionization and multiple reaction monitoring with an API 4000 Qtrap. The assay was linear in plasma from 0.5 to 200 ng/ml and 2.5 to 1000 ng/ml in each bupropion and hydroxybupropion enantiomer, respectively. The assay was linear in urine from 5 to 2000 ng/ml and 25 to 10,000 ng/ml in each bupropion and hydroxybupropion enantiomer, respectively. Intra- and inter-day accuracy was >98% and intra- and inter-day coefficients of variations were less than 10% for all analytes and concentrations. The assay was applied to a subject dosed with racemic bupropion. The predominant enantiomers in both urine and plasma were (R)-bupropion and (R,R)-hydroxybupropion. This is the first LC-MS/MS assay to analyze the enantiomers of both bupropion and hydroxybupropion in plasma and urine.     

Determination of plasma concentrations of epirubicin and its metabolites by high-performance liquid chromatography during a 96-h infusion in cancer chemotherapy

In order to determine epirubicin and its metabolites at low concentrations (<38 ng/ml) in small plasma samples, a fast reliable method based on a precipitation pre-treatment and sensitive reversed-phase isocratic HPLC has been developed and validated for epirubicin in the range 5–100 ng/ml. The R.S.D. was 5–9% over this concentration range. For human serum containing 25 ng/ml of epirubicin, the inter- and intra-day variation was <10%. Recoveries of the metabolites epirubicinol, 7-deoxydoxorubicinone and 7-deoxydoxorubicinolone at 20 ng/ml ranged from 94–104%. The assay has been used to study human plasma samples taken during a 96-h infusion of epirubicin in a patient with multiple myeloma. The combined levels of the unseparated metabolites, epirubicin glucuronide and epirubicinol glucuronide, were semiquantitatively determined after treatment with β-glucuronidase. The metabolites epirubicinol and 7-deoxydoxorubicinolone, but not 7-deoxydoxorubicinone, were also detected and measured.     

Study on the Interaction of β-Cyclodextrin and Berberine Hydrochloride and Its Analytical Application

The fluorescence enhancement of berberine hydrochloride (BBH) as a result of complex with β-cyclodextrin (β-CD) is investigated. The mechanism of the inclusion was studied and discussed by spectrofluoremetry and infrared spectrograms. The results showed that a 1∶1 (β-CD: BBH) complex was formed with an apparent association constant of 4.23×10² L/mol. Based on the enhancement of the fluorescent intensity of berberine hydrochloride, a new spectrofluorimetric method for the determination of BBH in the presence of β-CD was developed. The linear range was 1.00∼4.00 µg/mL with the detection limit of 5.54 ng/mL. The proposed method was successfully applied to the determination of BBH in tablets.     

β-Cyclodextrin-threaded Biocleavable Polyrotaxanes Ameliorate Impaired Autophagic Flux in Niemann-Pick Type C Disease

Niemann-Pick type C (NPC) disease is characterized by the lysosomal accumulation of cholesterols and impaired autophagic flux due to the inhibited fusion of autophagosomes to lysosomes. We have recently developed β-cyclodextrin (β-CD)-threaded biocleavable polyrotaxanes (PRXs), which can release threaded β-CDs in response to intracellular environments as a therapeutic for NPC disease. The biocleavable PRXs exhibited effective cholesterol reduction ability and negligible toxic effect compared with hydroxypropyl-β-CD (HP-β-CD). In this study, we investigated the effect of biocleavable PRX and HP-β-CD on the impaired autophagy in NPC disease. The NPC patient-derived fibroblasts (NPC1 fibroblasts) showed an increase in the number of LC3-positive puncta compared with normal fibroblasts, even in the basal conditions; the HP-β-CD treatment markedly increased the number of LC3-positive puncta and the levels of p62 in NPC1 fibroblasts, indicating that autophagic flux was further perturbed. In sharp contrast, the biocleavable PRX reduced the number of LC3-positive puncta and the levels of p62 in NPC1 fibroblasts through an mTOR-independent mechanism. The mRFP-GFP-LC3 reporter gene expression experiments revealed that the biocleavable PRX facilitated the formation of autolysosomes to allow for autophagic protein degradation. Therefore, the β-CD-threaded biocleavable PRXs may be promising therapeutics for ameliorating not only cholesterol accumulation but also autophagy impairment in NPC disease.     

Cholesterol in cerebrospinal fluid of brain tumor patients

A sensitive method for assay of total cholesterol (free plus esterified) in one ml of CSF is presented. Patients with brain tumors showed much higher levels of this sterol in CSF than those with neoplasia external to the central nervous system. Repeated assay of CSF cholesterol in post-surgical follow-up of brain tumor patients undergoing chemotherapy may provide a biochemical tool for detection of renewed tumor activity.