Determination of imipramine, desipramine and their hydroxy metabolites by reversed-phase chromatography with ultraviolet and coulometric detection.

A reversed-phase high-performance liquid chromatographic method is described which analyzes imipramine, desipramine and their corresponding 2-hydroxy metabolites with sequential ultraviolet and coulometric detection from a single common extraction step, so that a wider dynamic range of plasma concentrations can be measured requiring smaller sample volumes. Applicability is broader including single-dose pharmacokinetic studies as well as steady-state concentrations. The extraction procedure gives excellent recoveries for imipramine, desipramine and their metabolites (mean +/- S.D.): ultraviolet detection, imipramine 99.5 +/- 0.68%, desipramine 100 +/- 0.0%, 2-hydroxyimipramine 97.8 +/- 3.5% and 2-hydroxydesipramine 93.1 +/- 4.22%; coulometric detection, imipramine 97.5 +/- 1.9%, desipramine 98.3 +/- 1.2%, 2-hydroxyimipramine 90.3 +/- 4.0% and 2-hydroxydesipramine 86.6 +/- 7.5%.     

Preparative HPLC for the Facile Isolation of Drug Glucuronide Conjugates from Crude Extracts of Urine

Abstract

Reverse-phase preparative HPLC has been used to advantage for the isolation of crystalline quantities of drug glucuronide conjugates from crude urinary extracts. Following chronic administration of large doses of diazepam, levorphanol and hydroxyethylflurazepam to dogs, urine was collected and the water soluble drug conjugates adsorbed on a column of Amberlite XAD-2 resin. In each instance elution with methanol and solvent evaporation yielded a crude oil (approx. 3 g) which was chromatographed in one portion on either PrepPAK 500 C₁₈ (Waters) or Magnum 40 ODS-3 (Whatman) columns using aqueous methanol solvent systems. A greater than 90% purification was achieved in this single initial chromatographic step. Employing a combination of subsequent semi-preparative HPLC steps on either C₁₈ or silica gel columns, milligram quantities of the glucuronides of oxazepam, levorphanol and hydroxyethylflurazepam were isolated. The isolation procedures provide a general approach for obtaining milligram quantities of intact drug conjugates which may otherwise be difficult to obtain by chemical synthesis. Such conjugates can be used as authentic standards in the quantitation of certain drug metabolites in biological media during pharmacokinetic/biopharmaceutic studies.     

High-performance liquid chromatography of proteins: purification of alpha-fetoprotein from fetal calf serum

High-performance liquid chromatography was utilized for the purification of bovine alpha-fetoprotein (BAFP) from fetal calf serum (FCS). An initial step in the purification involved absorption of charcoal delipidated FCS on Cibacron Blue F3GA gel. The Cibacron Blue pre-purified FCS was then chromatographed on a Polyanion SI weak anion-exchange column. The BAFP isolated had a purity of greater than 93% with an overall yield of 48% from FCS. The procedure was applicable for semi-preparative scale purification of BAFP.     

Purification of a basic glycoprotein allergen from pollen of timothy by high-performance liquid chromatography

The use of high-performance ion-exchange and size-exclusion chromatography in the purification of the basic timothy pollen allergen antigen 30 (Ag 30) was investigated. The most efficient purification was achieved when an initial purification step on a CM-Sepharose CL-6B column was followed by chromatography on Mono S and TSK G 2000 SW columns. This procedure was highly reproducible and well suited for semi-preparative scale purification of the allergen. The purified allergen gave one band on isoelectric focusing, corresponding to a pI of 9.30. On fused rocket immunoelectrophoresis a single precipitate was obtained that coincided with the allergenic activity.     

Practical reversed-phase high-performance liquid chromatography method for laboratory-scale purification of recombinant human thyrotropin

A small, semi-preparative C(4) RP-HPLC column was used to set up the conclusive laboratory-scale purification of Chinese hamster ovary-derived human thyrotropin (hTSH), after a preliminary concentration-purification of an extremely dilute and poorly ( approximately 0.6 microg hTSH/mL; mass fraction=0.35%) conditioned medium on a cation exchanger. Several fractions of this eluate were repeatedly injected on the semi-preparative column, obtaining, in a single run (<1h chromatographic time), a concentrated pool ( approximately 1.2 mg/mL) of highly purified hTSH that could be further concentrated to >3 mg/mL and then efficiently lyophilized. The overall recovery in the rapid RP-HPLC purification step, including concentration and lyophilization, was of the order of 80%. The final product, when tested via a precise, single-dose in vivo bioassay, confirmed that it did not suffer any loss of bioactivity. This same methodology can be easily adapted to the small-scale purification of other recombinant products, even when obtained from genetically modified organisms at extremely low concentrations and mass fractions.     

Preparative purification of leuprorelin by HPLC applying a “saw-tooth” gradient

Summary Recently we reported on the development of a new semipreparative HPLC technique for the purification of leuprorelin [1]. This new method, referred to as a “saw-tooth technique” utilizes two successive gradients of two different eluent systems on the same column. Here we show our new results on extending the purification of leuprorelin from the semi-preparative 0.1–2 g) to the preparative (3–25 g) scale. Presented at Balaton Symposium on High Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

Evaluation of the potential of surface enhancement Raman spectroscopy for detection of tricyclic psychotropic drugs. Case studies on imipramine and its metabolite

The potential use of surface Raman enhanced spectroscopy (SERS) for confirmatory identification and the semi-quantitative analysis of selected tricyclic antidepressants (TCAs) is examined utilizing a conventional silver colloid. Raman and SERS spectra of aqueous solutions of imipramine (Imi) and its metabolite, desipramine (Des), were recorded as the function of concentration using NIR excitation. A good linear correlation is observed for the dependence of the SERS signal at 684 cm(-1) (R(2) = 0.9997) on Imi concentration over the range of 0.75-7.5 μM. The limit of detection of imipramine in the silver colloidal solution is 0.98 μM. SERS spectra of Imi and Des were also recorded for blood plasma samples without prior purification as well as after the use of standard solid phase extraction. All spectra show the characteristic spectral profile of the molecules and moreover, stronger signal enhancement is observed for Imi in the "raw" samples as opposed to Imi extracted from a biological matrix.     

Comparison of analytical and semi-preparative columns for high-performance liquid chromatography-solid-phase extraction-nuclear magnetic resonance

The application of analytical and semi-preparative columns in reversed-phase liquid chromatography-solid-phase extraction-nuclear magnetic resonance (HPLC-SPE-NMR) was compared. The work was aiming at separating a higher sample amount in a single run and in this way to reduce the necessary NMR measurement time of separated compounds. Several parameters for compound separation and trapping procedures were optimised: flow rate of HPLC and make-up water pumps, choice of stationary phase cartridges and drying time. The separation and loadability of nine model compounds on analytical and semi-preparative columns was determined, as well as the focussing capacity of SH-type SPE cartridges. It was found that a semi-preparative column--or multiple peak trapping on analytical columns--gave better results than a standard 4.6mm analytical column for non-polar compounds (e.g. flavonoid aglycones, sesquiterpene lactones, non-polar terpenes, logP>2), but for polar compounds (logP<-2) did not offer any advantage over an analytical column, or was even disadvantageous. For intermediately polar compounds (-2相似文献   

Purification of a 166mHo solution by successive high-performance liquid chromatography and gravitational chromatography for half-life determination

A methodology to purify a ^166mHo solution has been developed by a combination of activity and mass concentration measurements in order to further determine the ^166mHo half-life. The isobaric interference at m/q ? 166 requires Ho purification from non-natural Er with a high purification degree due to the large amount of Ho as opposed to Er. The Ho/Er separation was achieved using high-performance liquid chromatography on a semi-preparative column followed by purification on gravitational chromatography. The efficiency of the separation was evaluated after precise determination of the Er isotopic composition. The purification methodology enabled to separate Ho from Er.     

Liquid chromatography analysis of monosubstituted sulfobutyl ether-beta-cyclodextrin isomers on porous graphitic carbon

The retention behaviour of the three positional isomers of monosubstituted sulfobutyl ether-beta-cyclodextrin was investigated on a porous graphitic carbon (PGC) column. The influence of the mobile phase composition (nature and concentration of organic and electronic modifiers) was studied as well as the effect of column temperature. These hydrophilic and anionic analytes were highly retained on the PGC stationary phase compared to octadecyl bonded phases. The retention is mainly governed by a reversed-phase mechanism with electronic interaction playing a secondary role. An increase in solute retention and efficiency with temperature was observed. Successful isocratic separation with satisfactory baseline resolution of the three isomers of monosubstituted sulfobutyl ether-beta-cyclodextrin was achieved at 75 degrees C on a Hypercarb column by using ammonium acetate as electronic modifier in water-acetonitrile (83:17). The chromatographic methodology developed can be easily used for relative quantification of each isomer within a mixture and can be applied for semi-preparative purification of each one. The evaporative light scattering detector allows the detection of these non UV-visible absorbing molecules.     

Pharmacokinetics and excretion study of bergenin and its phase II metabolite in rats by liquid chromatography tandem mass spectrometry

A sensitive and selective liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method for the determination of bergenin and its phase II metabolite in rat plasma, bile and urine has been developed. Biological samples were pretreated with protein precipitation extraction procedure and enzymatic hydrolysis method was used for converting glucuronide metabolite to its free form bergenin. Detection and quantitation were performed by MS/MS using electrospray ionization and multiple reaction monitoring. Negative electrospray ionization was employed as the ionization source. Sulfamethoxazole was used as the internal standard. The separation was performed on a reverse‐phase C₁₈ (250 × 4.6 mm, 5 μm) column with gradient elution consisting of methanol and 0.5% aqueous formic acid. The concentrations of bergenin in all biological samples were in accordance with the requirements of validation of the method. After oral administration of 12 mg/kg of the prototype drug, bergenin and its glucuronide metabolite were determined in plasma, bile and urine. Bergenin in bile was completely excreted in 24 h, and the main excreted amount of bergenin was 97.67% in the first 12 h. The drug recovery in bile within 24 h was 8.97%. In urine, the main excreted amount of bergenin was 95.69% in the first 24 h, and the drug recovery within 24 h was <22.34%. Total recovery of bergenin and its glucuronide metabolite was about 52.51% (20.31% in bile within 24 h, 32.20% in urine within 48 h). The validated method was successfully applied to pharmacokinetic and excretion studies of bergenin.     

Separation of 50 S ribosomal proteins from sulfolobus acidocaldarius by discontinuous reversed-phase chromatography

Summary The total protein of the 50S ribosomal subunit (TP50) from the archaebacterium Sulfolobus acidocaldarius was prefractionated by discontinuous reversed-phase HPLC with several column combinations. The purity of the eluted fractions was tested by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional PAGE (2D-PAGE). With regard to the load capacity and selectivity, best results were obtained with a semi-preparative three-column combination (total length 67.5cm). Twelve A₂₃₀ units of TP50 were separated into 35 fractions, 19 of which contained nearly pure proteins. When the sample load was increased to 176A₂₃₀ units (109mg), 13 of 28 collected fractions still contained nearly pure proteins. The selectivity of a semi-preparative short-column combination (total length 12cm) was similar to that of the semi-preparative column combination, and separation time could be reduced to one third of that required for the longer column combination. The load capacity of the short-column combination was lower than that of the semi-preparative column combination.     

Fast-atom bombardment and thermospray mass spectrometry for the characterization of two glucuronide metabolites of methapyrilene

Two conjugated metabolites of methapyrilene hydrochloride isolated from mouse-hepatocytes were examined by mass spectrometry using fast-atom bombardment (FAB) and thermospray ionization. The major metabolite, methapyrilene glucuronide, was identified based on a prominent peak due to the protonated molecule as well as the loss of the dimethylamine and sugar moieties. Identification of the second metabolite was complicated by large signals associated with the biological sample matrix. The complementary nature of the fragmentation observed in the mass spectra using FAB and thermospray ionization allowed this metabolite to be identified as the desmethylmethapyrilene glucuronide. The fragmentation observed using FAB ionization was not greatly affected by the presence of the glucuronide moiety. While loss of the sugar moiety indicated a glucuronide, additional fragmentation confirmed the presence of the underlying ethylenediamine substructure which is characteristic of this class of antihistamines.     

Isolation of three glucaric acids from Leonurus japonicus Houtt. by using high-speed countercurrent chromatography combined with semi-preparative high-performance liquid chromatography

The isomerism of glucaric acids and the complexity of the composition of Leonurus japonicus Houtt. increased the difficulty of the separation of glucaric acids from the herb. In the present study, three glucaric acids were isolated from Leonurus japonicus Houtt. by using high-speed countercurrent chromatography combined with semi-preparative high-performance liquid chromatography. Cation exchange resin chromatography was applied to remove the alkaloids and enrich the glucaric acid fractions. Preliminary separation of the glucaric acid extract by high-speed countercurrent chromatography was carried out at 45℃ by using an optimized solvent system of ethyl acetate/n-butanol/formic acid/water (1:1:0.01:2, v/v/v/v) with satisfied stationary phase retention and separation factor. The semi-preparative high-performance liquid chromatography was used for further separation and purification of the target fractions, and three monomeric compounds were obtained with purities of 90.0, 91.0, and 95.3%. UV spectroscopy, NMR spectroscopy, and mass spectrometry were employed to identify their structures, which were assigned as 2-syringyl glucaric acid, 2,4-disyringyl glucaric acid, and 3,4-disyringyl glucaric acid, respectively, and 2,4-disyringyl glucaric acid was reported for the first time.     

Preparative reversed-phase high-performance liquid chromatography collection efficiency for an antimicrobial peptide on columns of varying diameters (1mm to 9.4mm I.D.)

The present study examines the effect of reversed-phase high-performance liquid chromatography (RP-HPLC) column diameter (1mm to 9.4mm I.D.) on the one-step slow gradient preparative purification of a 26-residue synthetic antimicrobial peptide. When taken together, the semi-preparative column (9.4mm I.D.) provided the highest yields of purified product (an average of 90.7% recovery from hydrophilic and hydrophobic impurities) over a wide range of sample load (0.75-200mg). Columns with smaller diameters, such as narrowbore columns (150x2.1mm I.D.) and microbore columns (150x1.0mm I.D.), can be employed to purify peptides with reasonable recovery of purified product but the range of the crude peptide that can be applied to the column is limited. In addition, the smaller diameter columns require more extensive fraction analysis to locate the fractions of pure product than the larger diameter column with the same load. Our results show the excellent potential of the one-step slow gradient preparative protocol as a universal method for purification of synthetic peptides.     

An unusual sesquiterpenoid from Hypochaeris achyrophorus (Asteraceae)

The novel sesquiterpenoid 8alpha-hydroxyhypoglabric acid (1) was isolated from a methanolic extract of whole plants of Hypochaeris achyrophorus L. The compound was isolated by silica gel column chromatography (CC), repeated Sephadex LH-20 CC, and semi-preparative RP-HPLC. Structure elucidation was accomplished by high resolution mass spectrometry and by 1D- and 2D-NMR spectroscopy. The chemosystematic significance of the new compound is discussed in the context of sesquiterpenoids from other members of the Hypochaeridinae and in the light of recent molecular data on the phylogeny of this group.     

Quantitative determination of buprenorphine,naloxone and their metabolites in rat plasma using hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry

A rapid and sensitive LC–MS/MS method was developed and validated for the simultaneous determination of buprenorphine and its three metabolites (buprenorphine glucuronide, norbuprenorphine and norbuprenorphine glucuronide) as well as naloxone and its metabolite naloxone glucuronide in the rat plasma. A hydrophilic interaction chromatography column and a mobile phase containing acetonitrile and ammonium formate buffer (pH 3.5) were used for the chromatographic separation. Mass spectrometric detection was achieved by an electrospray ionization source in the positive mode coupled to a triple quadrupole mass analyzer. The calibration curves for the six analytes displayed good linearity over the concentration range 1.0 or 5.0–1000 ng/mL. The intra and inter‐day precision (CV) ranged from 2.68 to 16.4% and from 9.02 to 14.5%, respectively. The intra‐ and inter‐day accuracy (bias) ranged from −14.2 to 15.2% and from −9.00 to 4.80%, respectively. The extraction recoveries for all the analytes ranged from 55 to 86.9%. The LC–MS/MS method was successfully applied to a pharmacokinetic study of buprenorphine–naloxone combination in rats.     

Therapeutic monitoring of imipramine and desipramine by micellar liquid chromatography with direct injection and electrochemical detection

A micellar liquid chromatographic (MLC) procedure was developed for the clinical monitoring of imipramine and its active metabolite, desipramine. The determination of these highly hydrophobic substances was carried out after direct injection of the serum samples using a mobile phase composed of 0.15 m SDS--6% (v/v) pentanol buffered at pH 7, pumped at 1.5 mL/min into a C(18) column (250 x 4.6 mm), and electrochemical detection at 650 mV. Using this MLC method, calibration was linear (r > 0.995) and the limits of detection (ng/mL) were 0.34 and 0.24 for imipramine and desipramine, respectively. Repeatabilities and intermediate precision were tested at three different concentrations in the calibration range and a CV (%) below 2.2 was obtained. In this MLC procedure, the serum is determined without treatment, thus allowing repeated serial injections without changes in retention factors, and reducing the time and consumables required to carry out the pretreatment process. The assay method can be applied to the routine determination of serum imipramine and its metabolite in therapeutic drug monitoring.     

Isolation and purification of osthole and imperatorin from Fructus Cnidii by semi-preparative supercritical fluid chromatography

Fructus Cnidii, the dried ripe fruit of Cnidium monnieri (L.) Cusson., has been widely used in traditional Chinese medicine. Osthole and imperatorin are the main active ingredients of Fructus Cnidii and had been found of antispasmodic, anti-HIV, anti-fungal, anti-viral, anti-tumor, anti-mutagenic, anti-arrhythmic, hypotensive, and broad-spectrum antimicrobial effects. A supercritical fluid chromatography (SFC) method for isolation and purification of osthole and imperatorin from Fructus Cnidii was established in this work. The separation conditions, including the stationary phase, the organic modifier, the composition and the flow rate of the mobile phase, column backpressure and column temperature, were optimized on analytical scale at first. And then a semi-preparative SFC (SP-SFC) method was developed based on the conditions of analytical scale SFC. SP-SFC was accomplished on YMC-Pack NH₂ column. Ethanol was used as the modifier and its percentage in the mobile phase was 3%. The flow rate of the mobile phase was 20?mL/min, column backpressure was 13?MPa, column temperature was 318?K, detection wavelength was 310?nm, and injection volume was 0.2?mL. Under the optimum conditions, osthole and imperatorin were obtained with high purities as determined by high performance liquid chromatography. The chemical structures of the obtained compounds were identified by nuclear magnetic resonance and mass spectrum analysis.     

System to screen and purify active ingredients from herbal medicines using hydrogel-modified human umbilical vein endothelial cell membrane chromatography coupled with semi-preparative high-performance liquid chromatography-offline-high-performance liquid chromatography-mass spectrometry

Analytical screening and validation systems based on a combination of cell membrane chromatography and two-dimensional chromatography-tandem mass spectrometry are incapable of providing prepared samples containing the active ingredients found in traditional Chinese medicine; therefore, these samples cannot be directly used in subsequent studies. In this study, a semi-preparative cell membrane chromatography column was developed using a hydrogel-modified carrier and human umbilical vein endothelial cells to optimize prepared conditions, such as hydrogel polymerization, cell fragmentation, and cell membrane volume. This increased the binding ratio of membrane protein and carrier to 15.79 mg/g. The column was systematically evaluated using multitarget tyrosine kinase inhibitors that displayed good specificity and reproducibility. Subsequently, using the column coupled with a semi-preparative high-performance liquid chromatography-offline-high-performance liquid chromatography-mass spectrometry system, 15 active ingredients were screened and purified from Indigo naturalis, and five main components were identified: l -lysine, oxyresveratrol, tryptanthrin, isorhamnetin, and indirubin. Furthermore, the pharmacological effects of the ingredients were confirmed using cell proliferation and apoptosis assays. Results revealed potent proliferation-inhibiting and apoptosis-promoting abilities on human chronic myelogenous leukemic cells and human promyelocytic leukemic cells (p < 0.001). Overall, the system presented screening and purification functions that could be used to prepare I. naturalis samples acting on the epidermal growth factor receptor and vascular endothelial cell growth factor.