Determination of fluvoxamine in rat plasma by HPLC with pre-column derivatization and fluorescence detection using 4-fluoro-7-nitro-2,1,3-benzoxadiazole

A sensitive, simple and reliable method using high-performance liquid chromatographic (HPLC) assay of fluvoxamine (FLU), a selective serotonin reuptake inhibitor (SSRI), in rat plasma after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was developed in this study. Extracted plasma samples were mixed with NBD-F at 60 degrees C for 5 min and injected into HPLC. Retention times of FLU and an internal standard (propafenone) derivative were 15.5 and 13.5 min, respectively. The calibration curve was linear over the range 0.015-1.5 microg/mL (r2 = 0.9985) and the lower limits of detection and quantification of FLU were 0.008 and 0.015 microg/mL, respectively, in 100 microL of plasma. The derivative sample was stable at 4 degrees C for 1 day. The coefficients of variation for intra-day and inter-day assay of FLU were less than 8.3 and 9.6%, respectively. Other SSRIs and centrally acting drugs did not interfere with the peak of the FLU derivative. The method was applied for analysis of the plasma samples from rats treated with FLU. These results indicate that the method presented is useful to determine the FLU levels in rat plasma of volumes as small as 100 microL and can be applied to pharmacokinetic studies.     

Simultaneous liquid chromatographic assay of amantadine and its four related compounds in phosphate-buffered saline using 4-fluoro-7-nitro-2,1,3-benzoxadiazole as a fluorescent derivatization reagent

Simultaneous HPLC assay of 1-adamantanamine hydrochloride (amantadine) and its four related compounds [2-adamantanamine hydrochloride (2-ADA), 1-adamantanmethylamine (ADAMA), 1-(1-adamantyl)ethylamine hydrochloride (rimantadine) and 3,5-dimethyl-1-adamantanamine hydrochloride (memantine)] in phosphate-buffered saline (pH 7.4) after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was developed. Phosphate-buffered saline samples were mixed with borate buffer and NBD-F solution in acetonitrile at 60 degrees C for 5 min and injected into HPLC. Five derivatives were well separated from each other. The lower limits of detection of amantadine, 2-ADA, ADAMA, rimantadine and memantine were 0.008, 0.001, 0.0008, 0.0015 and 0.01 microg/mL, respectively. The coefficients of variation for intra- and inter-day assay were less than 6.4 and 8.2%, respectively. The method presented was applied to a binding study of these compounds to human alpha(1)-acid glycoprotein. While affinity constants and capacities for ADAMA, rimantadine and memantine were calculated by means of Scatchard plots, those for the others were not determined. ADAMA, rimantadine and memantine were bound with different affinities and capacities. These results indicate that NBD-F is a good candidate as a fluorescent reagent to simultaneously determine amantadine and its four related compounds by HPLC after pre-column derivatization. Our method can be applied to binding studies for protein.     

Comparison of SSI with APCI as an interface of HPLC-mass spectrometry for analysis of a drug and its metabolites

Sonic spray ionization (SSI) was compared with atmospheric pressure chemical ionization (APCI) as an interface of high-performance liquid chromatography (HPLC)-mass spectrometry (MS) for sensitive analyses of a neuroleptic drug, haloperidol and its two metabolites, such as reduced haloperidol and 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP), in biological samples. For both SSI and APCI interfaces, HPLC-MS-MS gave higher sensitivity than HPLC-MS. The sensitivities by HPLC-SSI-MS-MS for haloperidol and reduced haloperidol were 100 and 30 times higher, respectively, than those by HPLC-APCI-MS-MS; no spectrum with recognizable peaks was obtained for CPHP with the APCI interface. Therefore, detection limits and regression equations were examined by the HPLC-SSI-MS-MS for human plasma and urine samples spiked with the above drug and its metabolites. Haloperidol, reduced haloperidol, and CPHP showed good linearity in the ranges of 5-800, 10-800, and 100-800 ng/mL, respectively, for both human plasma and urine; their detection limits were 2.5, 5, and 75 ng/mL, respectively, using a new polymer HPLC column which enabled direct application of biological samples.     

Improved conditions for the application of solid phase microextraction prior to HPLC-FLD analysis of anatoxin-a

Solid phase microextraction coupled with high performance liquid chromatography with fluorescence detection has been optimized and evaluated for a simple, rapid, and selective analysis of anatoxin-a. Four kinds of fiber (100 microm polydimethylsiloxane, 60 microm polydimethylsiloxane/divinylbenzene, 50 microm Carbowax/templated resin-100, and 85 microm polyacrylate) were evaluated for an efficient extraction of the toxin. Parameters relating to the desorption step, such as desorption mode, solvent composition, time for both static and dynamic desorption, as well as carryover, have been studied and optimized. The derivatization process was investigated using NBD-F as derivatizing reagent. Anatoxin-a derivative was formed when the anatoxin-a-loaded fiber was inserted in a vial containing 5 microL of NBD-F. Variables affecting extraction such us ionic strength, temperature, and time have been also optimized. The results obtained showed linearity in the range of 10-2000 ng and a limit of detection of 0.29 ng/mL in river water. The presented method has been applied to different environmental samples.     

Simultaneous determination of diethylene glycol and propylene glycol in pharmaceutical products by HPLC after precolumn derivatization with p-toluenesulfonyl isocyanate

A simple and reliable HPLC method was developed for the simultaneous quantitative analysis of diethylene glycol (DEG) and propylene glycol (PG) in pharmaceutical products by precolumn derivatization. The derivatization reagent p-toluenesulfonyl isocyanate (TSIC, 10 microL, 20% in ACN v/v) was added to 100 microL of the sample, and then 10 muL of water was added. The resulting derivatives were separated using a C(18)analytical column and a mobile phase composed of 0.01 M KH(2)PO(4)buffer (adjusted to pH 2.5 with phosphoric acid) and ACN (47:53 v/v) at 1 mL/min and 25 degrees C. For detection, UV light at 227 nm was used. The derivatization conditions including reaction time, temperature, and concentration of TSIC were optimized. The calibration curves were linear from 0.062 to 18.6 microg/mL (r(2)= 0.9999) and from 0.071 to 21.3 microg/mL (r(2) = 0.9999) for DEG and PG, respectively. The RSD values of intra- and interday assays were all below 4% for DEG and PG. The proposed method was then successfully applied to analyze two Armillarisin A injection samples and two spiked syrup samples.     

Determination of chlorophenols in water samples using simultaneous dispersive liquid-liquid microextraction and derivatization followed by gas chromatography-electron-capture detection

Simultaneous dispersive liquid-liquid microextraction (DLLME) and derivatization combined with gas chromatography-electron-capture detection (GC-ECD) was used to determine chlorophenols (CPs) in water sample. In this derivatization/extraction method, 500 microL acetone (disperser solvent) containing 10.0 microL chlorobenzene (extraction solvent) and 50 microL acetic anhydride (derivatization reagent) was rapidly injected by syringe in 5.00 mL aqueous sample containing CPs (analytes) and K(2)CO(3) (0.5%, w/v). Within a few seconds the analytes derivatized and extracted at the same time. After centrifugation, 0.50 microL of sedimented phase containing enriched analytes was determined by GC-ECD. Some effective parameters on derivatization and extraction, such as extraction and disperser solvent type and their volume, amount of derivatization reagent, derivatization and extraction time, salt addition and amount of K(2)CO(3) were studied and optimized. Under the optimum conditions, enrichment factors and recoveries are in the range of 287-906 and 28.7-90.6%, respectively. The calibration graphs are linear in the range of 0.02-400 microg L(-1) and limit of detections (LODs) are in the range of 0.010-2.0 microg L(-1). The relative standard deviations (RSDs, for 200 microg L(-1) of MCPs, 100 microg L(-1) of DCPs, 4.00 microg L(-1) of TCPs, 2.00 microg L(-1) of TeCPs and PCP in water) with and without using internal standard are in the range of 0.6-4.7% (n=7) and 1.7-7.1% (n=7), respectively. The relative recoveries of well, tap and river water samples which have been spiked with different levels of CPs are 91.6-104.7, 80.8-117.9 and 83.3-101.3%, respectively. The obtained results show that simultaneous DLLME and derivatization combined with GC-ECD is a fast simple method for the determination of CPs in water samples.     

Improved gas chromatography methods for micro-volume analysis of haloacetic acids in water and biological matrices

A fast headspace solid-phase microextraction gas chromatography method for micro-volume (0.1 mL) samples was optimized for the analysis of haloacetic acids (HAAs) in aqueous and biological samples. It includes liquid-liquid microextraction (LLME), derivatization of the acids to their methyl esters using sulfuric acid and methanol after evaporation, followed by headspace solid-phase microextraction with gas chromatography and electron capture detection (SPME-GC-ECD). The derivatization procedure was optimized to achieve maximum sensitivity using the following conditions: esterification for 20 min at 80 degrees C in 10 microL methanol, 10 microL sulfuric acid and 0.1 g anhydrous sodium sulfate. Multi-point standard addition method was used to determine the effect of the sample matrix by comparing with internal standard method. It was shown that the effect of the matrix for urine and blood samples in this method is insignificant. The method detection limits are in the range of 1 microg L(-1) for most of the HAAs, except for monobromoacetic acid (MBAA) (3 microg L(-1)) and for monochloroacetic acid (MCAA) (16 microg L(-1)). The optimized procedure was applied to the analysis of HAAs in water, urine and blood samples. All nine HAAs can be separated in < 13 min for biological samples and < 7 min for drinking water samples, with total sample preparation and analysis time < 50 min. Analytical uncertainty can increase dramatically as the sample volume decreases; however, similar precision was observed with our method using 0.1 mL samples as with a standard method using 40 mL samples.     

A simple and sensitive HPLC-fluorescence method for quantification of MDMA and MDA in blood with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a label

A sensitive high-performance liquid chromatographic method with fluorescence detection to determine 3,4-methylenedioxymethamphethamine (MDMA) and 3,4-methylenedioxyamphethamine (MDA) in human and rat whole blood or plasma samples was developed by using 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a label. MDMA and MDA in a small amount of blood sample (ca 100 microL) were extracted by liquid-liquid extraction with ethyl acetate, and were derivatized with DIB-Cl under mild conditions (10 min at room temperature). A good separation of DIB-derivatives could be achieved within 45 min using a commercially available ODS column with an isocratic eluent of 10 mM citric acid-20 mM Na(2)HPO(4) aqueous buffer (pH 4.0)-CH(3)CN-CH(3)OH (50:45:5, v/v/v %). The calibration curves prepared with 1-methyl-3-phenylpropylamine (MPPA) as an internal standard showed good linearity (r = 0.999) with 0.36-0.83 ng/mL detection limit at a signal-to-noise ratio of 3. MDMA and MDA in rat whole blood could be monitored for 6 h after a single administration of MDMA (2.2 mg/kg, i.p.). The pharmacokinetic parameters for MDMA and MDA obtained by triplicate measurements were 426 +/- 23 and 39 +/- 6 ng/mL (C(max)), 20 +/- 5 and 100 +/- 10 min (T(max)), respectively.     

Automated derivatization and analysis of malondialdehyde using column switching sample preparation HPLC with fluorescence detection

Analyte derivatization is advantageous for the analysis of malondialdehyde (MDA) as a biomarker of oxidative stress in biological samples. Conventionally, however, derivatization is time consuming, error-prone and has limited options for automation. We have addressed these challenges for the solid phase analytical derivatization of MDA from small volume tissue homogenate samples. A manual derivatization method was first developed using Amberlite XAD-2 (12 mg) as the solid phase. Subsequently an automated column switching process was developed that provided simultaneous derivatization and extraction of the MDA-DH hydrazone product on a cartridge packed with XAD-2, followed by quantitative elution of the product to an analytical LC column (Waters NovoPak C18, 3.9 x 150 mm). The LOD was 0.02 microg/mL and recovery was quantitative. The method was linear (r(2) >0.999) with precision < 5% from the LOQ (0.06 microg/mL) to at least 35 microg/mL. The method was successfully applied to the analysis of small volume (30 microL) mouse tissue homogenate samples. Endogenous levels of MDA in the tissues ranged from 20 to 40 nmol/g tissue (ca. 0.1-0.2 microg/mL homogenate). Compared to conventional MDA analyses, the current method has advantages in automation, selectivity, precision and sensitivity for analysis from very small sample volumes.     

Analysis of dideoxyadenosine triphosphate by CE with fluorescence detection. I. Derivatization through the phosphate group

A CZE method was developed, which separates 2',3'-dideoxyadenosine-5'-triphosphate (ddATP) from other metabolites and endogenous nucleotides at high concentrations (20-200 microg/mL) to allow UV detection. To enhance sensitivity, fluorescence detection which requires prior derivatization of compounds was examined. Precapillary derivatization of ddATP in the presence of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC) with dansyl ethylenediamine (dansyl EDA) was faster and stable compared to that of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylenediamine (BODIPY FL EDA). Reaction conditions, reagent concentrations and detection parameters were optimized and highest derivatization efficiency was achieved in 0.1 M 1-methylimidazole buffer (pH 8.0) with 140 mM EDAC in 1-methylimidazole buffer and 30 mM dansyl EDA in DMF for 90 min at 60 degrees C. Dansyl EDA derivatives of ddATP, 2'-deoxyadenosine-5'-triphosphate (dATP) and ATP were comigrating with the CZE method; therefore, a MEKC method was developed and optimized for repeatable separations. Upon dansylation, sensitivity of ddATP with fluorescence detection (LOQ = 12 ng/mL) was 160 times higher than UV detection (LOQ = 1.9 microg/mL).     

Quantitative analysis of betulinic acid in mouse,rat and dog plasma using electrospray liquid chromatography/mass spectrometry

Betulinic acid is under development as a therapeutic agent for the treatment of metastatic malignant melanoma. In support of pharmacokinetic and toxicological evaluations, a robust assay based on liquid chromatography/mass spectrometry (LC/MS) was developed for the quantitative analysis of betulinic acid. Sample preparation consisted of deproteinization of the plasma by the addition of three volumes of acetonitrile and one volume of methanol followed by centrifugation. Aliquots of the supernatant were analyzed using an isocratic reversed-phase high-performance liquid chromatography (HPLC) system coupled to a negative ion electrospray mass spectrometer. Deprotonated molecules of betulinic acid and the isomeric internal standard oleanolic acid were detected using selected ion monitoring at m/z 455. The limit of detection of betulinic acid was 0.5 pg (1.1 fM) injected on-column (50 pg/mL, 10 microL injection volume), and the limit of quantitation was 2 pg (4.4 fM, 200 pg/mL, 10 microL injection volume). Betulinic acid was stable in plasma samples at -20 degrees C for at least 3 weeks. The intra-day and inter-day coefficients of variation of the assay were < or =6.4 and < or =9.0%, respectively. The utility of the assay was demonstrated by analyzing betulinic acid spiked into mouse, rat and dog plasma, by determining the extent of binding of betulinic acid to plasma proteins, and by measuring betulinic acid in mouse and rat plasma following intraperitoneal or intravenous administration in vivo. At 15 and 25 microg/mL in mouse, rat or dog plasma, betulinic acid was 99.99% bound to serum proteins, and, at 5 microg/mL, betulinic acid was > or =99.97% bound.     

以睾酮为探针采用高效液相色谱法测定细胞色素CYP450 3A4的酶活性

建立了一种快速、高效的以睾酮作为探针药物评价细胞色素P450 3A4(CYP3A4)酶活性的高效液相色谱-紫外检测方法。采用的色谱柱为Phenomenex C18柱(4.6 mm×150 mm,5 μm),梯度洗脱,流速1.0 mL/min,紫外检测波长245 nm,柱温30 ℃。睾酮与大鼠肝微粒体温孵后,过已活化好的C18固相萃取小柱,收集甲醇洗脱液,于37 ℃水浴中通N2吹干,用50%甲醇复溶后进样分析测定。研究结果表明,6β-羟基睾酮的 保留时间为11.60 min,线性范围为0.5～32 μg/mL,最低检出质量浓度为0.02 μg/mL,提取率为88.41%～92.73%,方法的回收率为99.07%～101.30%;睾酮的保留时间为19.27 min,线性范围为0.5～40 μg/mL,最低检出质量浓度为0.01 μg/mL,提取率为89.59%～92.66%,方法的回收率为96.50%～98.03%。两者的日内、日间相对标准偏差均小于10%,温孵体系中的其他内源性物质不干扰测定。该方法快速、稳定、灵敏度高,适合体外睾酮及其代谢物6β-羟基睾酮的测定,可应用于体外CYP3A4酶活性的评价及酶动力学的研究。     

A fully automated amino acid analyzer using NBD-F as a fluorescent derivatization reagent

A fully automated amino acid analyzer using NBD-F (4- fluoro-7-nitro-2,1,3-benzoxadiazole) as a fluorescent derivatization reagent was developed. The whole analytical process was fully automated from derivatization, injection to HPLC separation and quantitation. The derivatization reaction conditions were re-evaluated and optimized. Amino acids were derivatized by NBD-F for 40 min at room temperature in the borate buffer (pH 9.5). The derivatives were separated within 100 min and fluorometrically detected at 540 nm with excitation at 470 nm. The detection limits for amino acids were in the range of 2.8-20 fmol. The calibration curves were linear over the range of 20 fmol to 20 pmol on column with the correlation coefficients of 0.999. The coefficients of variation were less than 5% at 3 pmol injection for all amino acids. Amino acids in rat plasma were determined by the proposed HPLC method.     

Use of a continuous flow solid-phase spectroscopic sensor using two sensing zones: determination of thiamine and ascorbic acid

A simple, rapid, inexpensive, and automated flow-through solid-phase spectroscopic sensing device is proposed for the sequential determination of 2 vitamins: thiamine and ascorbic acid. The vitamins are concentrated on ion-exchange gels, thiamine on Sephadex SP C-25, and ascorbic acid on Sephadex QAE A-25; both solid supports are packed in 2 different flow cells. The absorbance is monitored directly on the solid phase with a double-beam spectrophotometer at 250 nm, without derivatization or additional elution. With the use of 2 carrier/self-eluting solutions (0.1 5M sodium acetate/acetic acid and 0.18M citric acid/K2HPO4) and a sample volume of 1000 microL, the sensor responds linearly in the range of 0.5-15 and 3-50 microg/mL with detection limits of 0.14 and 0.36 microg/mL for thiamine and ascorbic acid, respectively. When the method was applied to synthetic samples and pharmaceutical preparations, precise and accurate values were obtained.     

Enhancing capillary liquid chromatography/tandem mass spectrometry of biogenic amines by pre-column derivatization with 7-fluoro-4-nitrobenzoxadiazole

This paper describes a capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS) determination of biogenic amines enhanced by pre-column derivatization with 7-fluoro-4-nitrobenzoxadiazole (NBD-F). Biogenic amines including tryptamine, N-methylsalsolinol, histamine, and agmatine were studied. The biogenic NBD-amine derivatives could be quantitatively enriched in-line on 20 x 0.25 mm capillary columns packed in-house with 5 microm C(8) silica particles. In an electrospray ionization (ESI) source these derivatives were ionized effectively, and collision-induced dissociation (CID) produced predominant characteristic ions allowing sensitive MS/MS detection. Agmatine, a potential neurotransmitter/modulator, was taken as a reference compound to study the analytical figures of merit of the procedure. The detection limit of agmatine was estimated to be 0.6 ng/mL (signal-to-noise (S/N) = 3). A linear calibration curve in the range 15-1000 ng/mL agmatine with an r value of 0.9997 was obtained. Tissue samples of rat brain, stomach, and intestine were analyzed. Minimum sample pre-treatment was needed. Each analysis was accomplished within ca. 12 min. The concentration of agmatine was found to be 0.246, 3.31, and 0.058 microg/g wet tissue in the brain, stomach, and intestine, respectively.     

Simultaneous analysis of haloperidol, its three metabolites and two other butyrophenone-type neuroleptics by high performance liquid chromatography with dual ultraviolet detection

We investigated simultaneous determination of haloperidol (HAL), its three metabolites [reduced HAL (R-HAL), 3-(4-fluorobenzoyl)propionic acid (FBPA) and 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP)] and two related compounds [spiperone (SPI) and droperidol (DRO)] in phosphate-buffered saline using high-performance liquid chromatography (HPLC) coupled with dual ultraviolet detection (220 and 250 nm). Retention times of HAL, R-HAL, FBPA, CPHP, SPI and DRO were 16.8, 11.8, 10.2, 4.1, 12.6 and 8.3 min, respectively. Their lower limits of detection were 7.5, 14, 4.5, 12, 10 and 20 ng/mL in the same order. The coefficients of variation for their intra- and inter-day assays were less than 7.8 and 9.4%, respectively. Of the other centrally acting drugs, only amoxapine interfered with the peak of DRO. Using our procedure, the binding study of tested compounds to synthetic melanin, human serum albumin and alpha1-acid glycoprotein was performed by determining the unbound concentration to total concentration ratio. These results indicated that simultaneous assay of HAL, R-HAL, FBPA, CPHP, SPI and DRO in phosphate-buffered saline by HPLC equipped with dual ultraviolet detection is simple, sensitive and reproducible. Also, our assay system can be applied to the binding study of these compounds to synthetic melanin, human serum albumin and alpha1-acid glycoprotein.     

Liquid chromatographic determination of polyamines in human urine based on intramolecular excimer-forming fluorescence derivatization using 4-(1-pyrene)butanoyl chloride

A liquid chromatographic method for highly sensitive and selective fluorometric determination of polyamines (putrescine, cadaverine, spermidine and spermine) in human urine is described. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butanoyl chloride (PBC), followed by reversed-phase liquid chromatography. The method offers higher sensitivity for determination of spermidine and spermine than previously reported method utilizing 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester as a derivatization reagent. Samples containing free polyamines in diluted human urine were directly derivatized with PBC and separated on an octyl column. The derivatives were detected at excitation 345 and emission 475 nm wavelengths. For determination of total polyamine content, the conjugated polyamines were first hydrolyzed in 4 M HCl. The detection limits (signal-to-noise ratio = 3) for polyamines in urine were 1.1-3.4 pmol/mL. At optimized derivatization and chromatographic conditions, interferences such as biogenic monoamines gave no peaks or the peaks did not interfere with the peaks of polyamine derivatives. In conclusion, the present derivatization method allows direct determination of polyamines in human urine samples without the need for sample clean-up procedures.     

Drop-to-drop solvent microextraction coupled with gas chromatography/mass spectrometry for rapid determination of trimeprazine in urine and blood of rats: application to pharmacokinetic studies

A simple and rapid method based on drop-to-drop solvent microextraction (DDSME) coupled with gas chromatography/mass spectrometry (GC/MS) has been successfully applied for the pharmacokinetic studies of trimeprazine in 8 microL of urine and blood samples of rats. Several factors that influenced the extraction efficiency of DDSME, such as selection of organic solvent, extraction time, exposure volume of organic phase, addition of salt and pH, were optimized. Linearity was obtained over the concentration ranges of 0.2-10, 0.25-7.0 and 0.5-6.0 microg/mL with correlation coefficients of 0.998, 0.996 and 0.993 in deionized water, urine and blood samples of rats, respectively. The limits of detection (LODs) of trimeprazine were 0.05, 0.06 and 0.1 microg/mL in deionized water, urine and blood samples. The concentrations of trimeprazine obtained in urine and blood samples of rats were 0.21-1.25 and 2.72-0.22 microg/mL, respectively, after a single intravenous administration of this drug. The enrichment factors and LOD values obtained by DDSME coupled to GC/MS were compared with those of hollow fiber liquid-phase microextraction (HF-LPME) combined with GC/MS. We believe that this novel approach can be very useful in clinical application since only one microdrop of biological samples was required to perform the pharmacokinetic studies from rats, so the sample pretreatments for animal experiments can be very easy too.     

Liquid chromatographic assay for the cyclic depsipeptide aplidine, a new marine antitumor drug, in whole blood using derivatization with trans-4'-hydrazino-2-stilbazole

A sensitive bio-analytical assay for the depsipeptide aplidine in plasma has been modified and tested for human whole blood samples. The adapted method is based on reversed-phase liquid chromatography and fluorescence detection of the trans-4'-hydrazino-2-stilbazole derivative of the analyte. Aplidine is isolated from the matrix by solid-phase extraction on an octadecyl modified silica stationary phase. After evaporation of the acetone eluate, the derivatization with the hydrazino reagent is performed in a water-acetonitrile mixture at pH = 4. The reaction mixture is injected directly into the chromatograph and the analyte is quantified by fluorescence detection at 410 and 560 nm for excitation and emission, respectively. The method has been validated in the 2-100 ng/mL range, with 2 ng/mL being the lower limit of quantification. Precision and accuracy both meet the current requirements for a bioanalytical assay. The stability of aplidine in whole blood at ambient temperature and at 37 degrees C is limited; recoveries in the range 60-85% were observed after 7 h. Further, adequate stability of aplidine in plasma at -80 and -20 degrees C for 35 months could now be demonstrated.     

Determination of paeoniflorin in rat plasma by a liquid chromatography-tandem mass spectrometry method coupled with solid-phase extraction

A rapid, sensitive and selective liquid chromatography-tandem spectrometry method was developed and validated for determination of paeoniflorin in rat plasma using geniposide as the internal standard. The samples were pretreated with solid-phase extraction using Extract-Clean cartridges. Separation of paeoniflorin and IS was achieved on a reversed-phase C18 column (50x4.6 mm i.d.) with a mobile phase made up of acetonitrile and 0.05% formic acid (25:75, v/v) at a flow rate of 0.5 mL/min. Detection was carried out on a triple quadrupole tandem mass spectrometer by multiple-reaction monitoring and an electrospray ionization source was employed as the ionization source. The lower limit of quantification obtained was 4 ng/mL (n=6) using 200 microL plasma with an accuracy of -3.67% (relative error) and a precision of 4.13% (relative standard deviation). A good linearity was found in the range of 4-1000 ng/mL. The intra- and inter-day relative standard deviations in the measurement of quality control samples 10, 150 and 800 ng/mL ranged from 3.73 to 4.94% and from 4.31 to 6.56%, respectively. The accuracy was from -3.93 to -1.11% in terms of relative error. The analyte and IS were stable in the battery of stability studies. This method was successfully applied to a pharmacokinetic study of paeoniflorin after a single oral administration of 53.36 mg/kg paeoniflorin to rats.