Simple and rapid high-performance liquid chromatographic method for the quantification of 3-methylhistidine

A procedure for the high-performance liquid chromatographic determination of tobramycin in serum is described using pre-column derivatisation with 1-fluoro-2,4-dinitrobenzene and subsequent chromatographic analysis on a reversed-phase column with ultraviolet detection. Gentamicin is used as the internal standard. The sensitivity is 0.5 mg/l with 50-μl samples. Precision, expressed as the coefficient of variation, is 3% or better in the concentration range 0.5–16 mg/l. The absolute recovery of tobramycin is 41%.The analyses of serum samples obtained in an in vivo experiment correlated well with the results from a microbiological assay. The influence of variation of derivatisation conditions and the implications for the reliability of the internal standardisation were studied. The 2,4-dinitrophenyl tobramycin derivative was synthesized and its structure was proved to be the fully derivatized tobramycin. Side-products of the derivatisation reaction were isolated.     

Determination of acyclovir by ultrafiltration and high-performance liquid chromatography

A simple, sensitive and rapid high-performance liquid chromatographic (HPLC) procedure to determine total serum acyclovir concentrations is described. The assay involves a heat inactivation step at 56°C to prevent risk of infection, ultrafiltration as a pretreatment step prior to ion-pair reversed-phase liquid chromatography using guanosine as internal standard, and ultraviolet detection at 254 nm. This method has excellent recovery (97–100%), linearity (0.5–100 mg/l) and precision (1.2–8.0% coefficient of variation). The detection limit is 50 μg/l. The assay proved to be suitable for therapeutic drug monitoring of acyclovir.     

Sensitive and specific determination of clindamycin in human serum and bone tissue applying liquid chromatography–atmospheric pressure chemical ionization–mass spectrometry

A method for the quantification of clindamycin in human serum and in human bone tissue samples applying high-performance liquid chromatography with atmospheric pressure chemical ionization–mass spectrometry (APCI–MS) is presented. Lincomycin is used as the internal standard. Serum samples are prepared only by protein precipitation with acetonitrile. Bone tissue samples have to be crushed and homogenized in extraction buffer prior to analysis. The chromatographic separation is achieved on an RP-18 stationary phase with 0.02% trifluoroacetic acid in water 60%/acetonitrile 40% v/v as mobile phase. The limits of quantification are 0.1 μg/ml for serum samples and 0.1 μg/g for bone tissue samples. The coefficients of variation for the assays are 4.48 and 8.41% at the limit of quantification for serum and bone tissue samples, respectively. Bone tissue samples as small as 50 mg can be used.     

Gas chromatographic method with mass-selective detection for the determination of 2-isopropoxyphenol in human urine

Human metabolism of the insecticide propoxur yields 2-isopropoxyphenol (IPP) which is excreted conjugated in urine. In this publication a sensitive and selective analytical method is described which permits the determination of IPP as a suitable parameter for biomonitoring. The clean-up of the hydrolysed urine samples consisted of steam distillation and solid-phase extraction using a reversed-phase column. IPP and the internal standard 2-ethoxyphenol were converted to their pentafluorobenzyl ethers. Excess of the derivatisation reagent was removed using deactivated silica gel. Separation and quantitative analysis was carried out by capillary gas chromatography and mass selective detection. Coefficients of variation were below 5% for concentrations from 6 to 300 μg/l. The detection limit was 0.5 μg/l. The method was checked by analysing six urine samples from pest controllers after indoor application of propoxur. The IPP concentrations ranged from 45 to 306 μg/g creatinine. IPP was not detected in urine specimens from 10 non-exposed persons. The sensitivity of the developed method permits the detection of latent exposure to propoxur.     

Determination of metformin in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of metformin, an oral antidiabetic agent, in plasma is described. Plasma samples containing the internal standard, phenformin, are eluted through Amprep extraction columns before injection into the chromatographic column, packed with μBondapak phenyl. The eluent is monitored at 236 nm. At a mobile phase flow-rate of 1.35 ml/min, the retention times of metformin and phenformin are 2.8 and 5.6 min, respectively. The intra-day coefficients of variation are 1.5 and 4.3% at metformin concentrations of 0.05 and 1 mg/l, respectively.     

A simple culture method for Brassica hypototyl protoplasts

Hypocotyl protoplasts from oil rape, Brassica napus L. cv. Isuzu were cultured in the dark at 25°C in a modified Nitsch and Nitsch medium containing 13% sucrose, 5 g/l Ficoll, 0.5 mg/l BAP, 1 mg/l NAA and 0.5 mg/l 2–4 D. Protoplasts floated on the surface of the medium and developed into microcolonies 0.5 mm in diameter in 4–6 weeks. The microcolonies also remained on the surface of the medium. Transfer to MS medium supplemented with 200 mg/l casein hydrolysate, 5mg/l BAP, 0.5 mg/l NAA and solidified with 0.6% agarose induced shoot regeneration in 3–4 weeks.Abbreviations BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - 2,4-D 2,4 — dichlorophenoxyacetic acid     

Production,maintenance and plant regeneration from cell suspension cultures of Stevia rebaudiana (Bert.) Bertoni

A method is described for producing and maintaining Stevia rebaudiana suspensions and regeneration of plants from calli derived from cell suspensions. Suspension cultures composed of isolated cells (ca. 10%) and cellular aggregates (5–100 cells) were obtained in 20–30 days by using friable callus as the initial inoculum in liquid medi with BA (0.5 mg/l)+2,4-D (1.0 mg/l), and periodic filtering (100–500 m sieves) with 6–7 days interval between subcultures. Cultures derived from actively growing calli are mainly diploid (2n=22) whereas those derived from senescent calli showed a wide variation in chromosome number (55–200). Stock cell suspensions which had been maintained for 3 years were plated on basal LS agar medium with BA (0.5 mg/l)+2,4D (0.5 mg/l) to form callus. Calli originating from predominantly 2n cell suspensions when transferred to medium with K (2.0 mg/l)+NAA (0.02 mg/l) were able to form buds. Shoot elongation and further rooting of isolated shoots was better on LS medium devoid of growth regulators. Variation in rooting capacity, plant vigour, morphological characters and chromosome number was found amongst regenerated plants.Abbreviations BA Benzylaminopurine - 2,4-D 2,4 - Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IAA Indoleacetic acid - IBA Indolebutyric acid - K Kinetin - LS Linsmaier & Skoog     

Development and validation of HPLC method for the determination of tobramycin in urine samples post-inhalation using pre-column derivatisation with fluorescein isothiocyanate

A reversed-phase liquid chromatography method involving pre-column derivatisation with fluorescein isothiocyanate (FITC, isomer I) for determination of tobramycin in urine samples after inhalation has been developed. FITC reacts with the primary amino groups of tobramycin and other aminoglycosides under mild conditions to form a highly fluorescent and stable derivative. The chromatographic separation was carried out on a Phenomenex Luna C(18) column at ambient temperature using a constant flow rate of 1 ml/min and mobile phase of acetonitrile-methanol-glacial acetic acid-water (420:60:5:515, v/v/v/v). The tobramycin-FITC derivative was monitored by fluorescent detection at an excitation wavelength 490 nm and emission wavelength 518 nm. The linearity of response for tobramycin was demonstrated at 11 different concentrations of tobramycin extracted from spiked urine, ranging from 0.25 to 20 microg/ml. Tobramycin and neomycin were extracted from spiked urine by a solid phase extraction clean-up procedure on a carboxypropyl-bonded phase (CBA) weak cation-exchange cartridge, and the relative recovery was >99% (n=5). The limit of detection (LOD) and limit of quantitation (LOQ) in urine were 70 and 250 ng/ml, respectively. The method had an accuracy of <0.2%, and intra-day and inter-day precision (in term of %coefficient of variation) were <4.89% and 8.25%, respectively. This assay was used for urinary pharmacokinetic studies to identify the relative lung deposition of tobramycin post-inhalation of tobramycin inhaled solution 300 mg/5 ml (TOBI) by different nebuliser systems.     

Micro-determination of clonazepam in plasma or serum by electron-capture gas—liquid chromatography

A rapid method is described for the electron-capture gas chromatographic determination of clonazepam in plasma or serum using methyl-clonazepam as an internal standard. The analysis is performed isothermally on the silicone stationary phase SP-2510DA (Supelco). With this liquid phase, gas chromatographic properties are comparable to methods involving acid hydrolysis or derivatisation. A short pre-column containing another phase is added to enhance resolution. The method involves a single extraction, requires 100 μl of sample and has a detection limit of 3 nmol/l. Response is linear at concentrations from 5–900 nmol/l and thus clonazepam analysis both during therapy and after overdosage is possible. Plasma and serum clonazepam levels are interchangeable.     

Somatic embryogenesis and plant regeneration from protoplasts of asparagus (Asparagus officinalis L.)

Protoplasts were isolated from embryogenic calli of Asparagus officinalis L. cv. Mary Washington and cultured in 1/2 MS medium with 1 mg/l NAA, 0.5 mg/l zeatin, 1 g/l L-glutamine, 0.6 M glucose and 0.1% Gellan Gum. Protoplasts started to divide after 3–4 d of culture and formed visible colonies after 30 d of culture. The percentage of colony formation (plating efficiency) was 7.2%. The colonies were then transferred onto Gellan Gum-solidified MS medium containing 1 mg/l 2,4-D and 3% sucrose for further growth. Somatic embryos were induced from all colonies of 0.5–1.0 mm size after transferring to 1/2 MS medium lacking growth regulators. After treating these somatic embryos (1–3 mm) in distilled water for a week, 30–40% of them germinated normally and grew into plantlets 20–30 d after transplanting on 1/2 MS medium containing 1 mg/l IBA, 1 mg/l GA₃ and 1% sucrose. These protoplast-derived plants were diploid with 20 chromosomes.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - GA₃ gibberellic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962)     

Protoplast culture and plant regeneration from Brassica carinata Braun

Protoplasts isolated from hypocotyls of three-day-old seedlings of Brassica carinata (Braun) cv R-2128 were cultured in a modified Nitsch and Nitsch liquid medium containing 13% sucrose, 0.4% Ficoll, 0.25 mg/l BA, 0.5 mg/l NAA and 0.5 mg/l 2,4-D. The density of medium caused the protoplasts and the developing microcalli to float on the surface of the liquid medium whereas all debris and lysed cells sank to the bottom of the culture plate. After 4–6 weeks developing microcalli were approximately 0.5 mm in diameter and were transferred onto MS medium containing 3% sucrose, 0.4% agarose, 200 mg/l casein hydrolysate, 5 mg/l BA and 0.5 mg/l NAA, pH 5.7. Approximately 20% of the calli transferred to this medium produced plantlets.Abbreviations BA 6-Benzylaminopurine - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashiqe-Skoog     

In vitro propagation through axillary bud multiplication in different mulberry genotypes

Axillary buds from 5 genotypes of mulberry belonging to 4 species were cultured on modified MS basal medium. A total of 30 media combinations were tried for all the genotypes. The response of axillary buds and the requirement for growth regulators varied with genotype. In Morus indica BAP (0.25–0.5 mg/l), and in M. alba and M. rotondifolia GA₃ (0.5–1.0 mg/l)were found to induce sprouting. Two genotypes of M. bombycis, namely Schimanochi and Mizusawa, developed healthy shoots on the incorporation of 2,4-D (0.5–1.0 mg/l) and BAP (0.5–2.0 mg/l), respectively. IBA (0.5 mg/l), along with cytokinin/auxin/gibberellin, had no effect on bud growth but helped root induction. Shoots developed from the axillary buds were further multiplied as nodal explants. MS basal medium supplemented with 0.5 mg/l IBA and LS vitamins was found best to produce healthy plantlets in all the genotypes. An average 89% survival was observed on transferring the plantlets to soil.Abbreviations MS Murashige and Skoog (1962) - LS Linsmaier and Skoog (1965) - IBA 3-indole-butyric acid - GA₃ Gibberellic acid - BAP 6-Benzylaminopurine - Kn Kinetin - 2,4-D 2,4-Dichlorophenoxyacetic acid     

Somatic embryogenesis and in vitro flowering of 3 species of bamboo

Plant regeneration via somatic embryogenesis was achieved in callus cultures derived from nodal explants of in vitro grown seedlings and excised mature zygotic embryos of three bamboo species on Murashige and Skoog's (MS) basal medium supplemented with 0.5 mg/l kinetin (Kn), 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 10 mg/l adenine sulphate (Ads) and 3% (w/v) sucrose incubated in the light or in the dark. Somatic embryos germinated (95–98%) into normal plants and were transferred to soil with 95% success. In vitro flowering was induced on shoots developed from nodal explants taken from somatic embryo regenerated plants of Bambusa vulgaris, Dendrocalamus giganteus and Dendrocalamus strictus on half-strength MS basal medium supplemented with 0.25 mg/l indole-3-butyric acid (IBA), 0.5 mg/l Ads, 0.5 mg/l gibberellic acid (GA₃) and 3% sucrose.Abbreviations BAP 6-benzylaminopurine - Kn kinetin - Ads adenine sulphate - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) basal medium - GA₃ gibberellic acid     

Plant regeneration from protoplasts isolated from callus ofGentiana crassicaulis

Fast growing calli induced from hypocotyl segments ofGentiana crassicaulis were used for preparation of protoplasts. High yields of viable protoplasts were produced in an enzyme solution containing 1–2% cellulase, I% pecfinase, and 0.5% Hemicellulase. Protoplasts were cultured in KM8P medium containing 1 mg/l 2,4-D, 0.5 mg/l 6BA, 500 mg/l LH, 0.5 M glucose and 0.1 M mannitol by the solid-liquid dual layer culture method. First division occurred within 4–5 days of culture at a frequency of 17.8%. Sustained divisions led to callus formation. Periodically diluting the cultures with freshly prepared liquid medium containing 1% glucose was critical for colony formation. Protocolonies about 2 mm in size were transferred onto MS medium supplemented with 3 mg/l ZT, 2 mg/l 6BA, 1 mg/l GA₃, 1 mg/l NAA and 6% sucrose to obtain embryogenic calli. Plantlets were regenerated via somatic embryogenesis at high frequency on hormone-free MS Medium.Abbreviations 6BA 6-benzylaminopurine - NAA naphthaleneacetic acid - 2,4-D 2,4 - dichlorophenoxyacetic acid - ZT zeatin - GA₃ gibberellic acid - LH lactalbumin hydrolysate - MES 2-(N-morpholino)-ethane sulfonic acid - MS Murashige & Skoog's medium(1962)     

Micro determination of gentamicin in serum by high-performance liquid chromatography with ultraviolet detection

A procedure for the high-performance liquid chromatographic determination of gentamicin in serum is described using pre-column derivatisation and UV detection. The serum proteins are precipitated with acetonitrile and the gentamicin components in the supernatant are derivatized with 1-fluoro-2,4-dinitrobenzene. The reaction products are chromatographed on a microparticulate C₁₈ reversed-phase column and detected at 365 nm. Sample volumes of 50 μl are sufficient for the determination of gentamicin concentrations in, and well below, the therapeutic range.     

Gas chromatographic analysis of therapeutic concentrations of maprotiline in serum, using flame-ionization detection

For the measurement of the tetracyclic antidepressant maprotiline in human serum, a gas chromatographic method with flame-ionization detection has been developed. The assay specifications obtained are as follows: a precision (C.V.) of 3.5–6.4%, and a relative recovery of 97–109% using amitriptyline as internal standard. The sensitivity of the assay from serum was 40 nmol/l. The applicability of the method has been shown by measuring steady-state serum levels of five inpatients. The steady-state serum levels of maprotiline given at a daily dosage of 75 mg varied from 272 to 570 nmol/l.     

Somatic embryogenesis and plant regeneration from immature inflorescence segments of Coix lacryma-jobi

Callus was obtained from segments of immature inflorescence of Coix lacryma-jobi cultured on N6 medium containing 1–2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 3–5% sucrose. Plantlets were regenerated when embryogenic calluses were transferred onto MS medium with 0.5 mg/l kinetin and 0.01 mg/l naphthaleneacetic acid (NAA). Regenerated plants had the diploid chromosome number (2n=20).     

Sensitive gas-liquid chromatographic method for the assay of the neuroleptic drug cis(Z)-flupentixol in human serum or plasma

A gas-liquid chromatographic (GLC) assay suitable for the analysis of the cis(Z)-stereoisomer of the antipsychotic drug flupentixol in human serum or plasma was developed. The minimal quantifiable concentration was 0.5 ng/ml and the day-to-day coefficient of variation was 11.2% at 1 ng/ml and 8.7% at 10 ng/ml. Following addition of perphenazine as the internal standard (I.S.) and aqueous NaOH, samples (2 ml) are extracted with n-hexane-isoamyl alcohol (98.5:1.5, v/v) (solvent), back-extracted to 0.1 M HCl and after one washing-step and addition of aqueous NaOH again extracted into 100 μl solvent. After evaporation to dryness, the extract is reconstituted in 20 μl solvent and evaporated to approximative 10 μl. A 4-μl aliquot is injected cool on-column onto the GLC system. A gas chromatograph HP 5890 with on-column injection port, nitrogen-phosphorus detector (NPD), a HP-1 25 m × 0.32 mm I.D., 0.5 μm capillary and hydrogen (3 ml/min, automated pressure control) as the carrier gas was applied. The negative influence of light on the assay was measured and discussed. The suitability of this method for clinical pharmacokinetic studies and therapeutic drug monitoring (TDM) was determined by the analysis of serum samples of 12 schizophrenic patients.     

Simultaneous determination of misonidazole and desmethylmisonidazole in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the simultaneous determination of misonidazole and desmethylmisonidazole in plasma is described. After plasma is deproteinized with methanol and the diluted supernatant is chromatographed on a C₁₈ reversed-phase column, both compounds are quantitated by means of an internal standard. The coefficients of variation of within-day and day-to-day precision are below 5.0% for misonidazole in the concentration range of 25–250 mg/l and below 6.1% for desmethylmisonidazole in the concentration range of 2.5–25.0 mg/l. Calibration curves are linear and an analytical recovery varying from 97.6 to 99.8% is obtained. The detection limits for misonidazole and desmethylmisonidazole in plasma are 1.4 mg/l and 0.7 mg/l, respectively.     

Shoot regeneration from stem and leaf callus of Eucalyptus tereticornis

Adventitious shoots were obtained from leaf and stem callus of Eucalyptus tereticornis SM. Callus was induced on B5 medium with 0.1 mg/l benzyladenine (BA) and 3 or 5 mg/l naphthalene acetic acid in the dark. Shoot initiation occurred on modified Woody Plant medium (mWP) containing 0.5 mg/l BA, 500 mg/l polyvinylpyrrolidone and 10% (v/v) coconut milk. Multiple shoots were also regenerated directly from hypocotyl segments of 4 to 6 week old seedlings on B5 medium with 0.5 mg/l BA. Regenerated shoots could be rooted with 100% efficiency on mWP medium containing 0.5 mg/l indolebutyric acid and transferred to soil in the greenhouse. Suspension cultures were obtained from the callus using B5 medium with 0.5 mg/l 2,4-dichlorophenoxyacetic acid. Callus clumps grew from less than 1 mm to 4–6 mm in diameter within two weeks on transfer to shoot regeneration medium but failed to form shoots or somatic embryos.Abbreviations BA Benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA Indolebutyric acid - NAA Naphthaleneacetic acid - PVP Polyvinylpyrrolidone - mWP modified Woody Plant medium Scientific Contribution No. 1689 from New Hampshire Agricultural Experiment Station.