Dansyl Chloride Derivatization Research Articles

Identification and quantification of S-allyl-l-cysteine in heated garlic juice by HPLC with ultraviolet and mass spectrometry detection

This paper presents a new high performance liquid chromatography (HPLC) method for the identification and quantification of S-allyl-l-cysteine (SAC), which is the most important bioactive compound present in heated garlic juice (HGJ). This method uses LC combined with an ultraviolet (LC–UV) and mass spectrometry detection (LC–MS/MS) method and is based on the dansyl chloride derivatization step. The method validation included selectivity, linearity, precision, accuracy, and sensitivity. The linear correlation coefficients (r) were always greater than 0.998. Intra- and inter-day precision for SAC at three concentrations demonstrated a relative standard deviation (RSD) of less than 15.3% for both UV and MS methods. The limit of quantification (LOQ) was 0.71 μg/g and 0.07 μg/g for UV and MS methods, respectively. The results were not significantly different between the SAC contents determined by the UV and MS detection in real HGJ samples. Both methods are proposed as useful quality control tools for accurate determination of SAC content of garlic preparations.

Quantification of β‐aminopropionitrile, an inhibitor of lysyl oxidase activity, in plasma and tumor of mice by liquid chromatography tandem mass spectrometry

Lysyl oxidase enzymes are reported to be involved in patho-physiological process such as tumorigenesis. β-Aminopropionitrile (BAPN) is an irreversible inhibitor of lysyl oxidase activity, suggesting a potentially useful therapeutic of interest in oncology. This paper describes the first assay concerning the quantification of BAPN by mass spectrometry. A high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) assay was developed for the quantification of BAPN in plasma and tumor of mice. This method combines dansyl chloride (Dns) derivatization and extraction using a solid-phase extraction Oasis Max column. Deuterated BAPN was used as internal standard (IS). Separation was achieved using an C₁₈ column HypersylGold, (ThermoElectron), 3.0 µm (100 × 2.1 mm i.d.). Gradient elution with water containing 0.1% acetic acid (A) and acetonitrile containing 0.1% acetic acid (B) was applied. Detection was performed with an electrospray ionization interface operating in negative ion mode. Selected reaction monitoring was used with ion transitions m/z 302 → 249 for BAPN-Dns and m/z 306 → 250 for the IS. The method was fully validated in plasma and was linear and sensitive in the range of 10-500 ng/mL. The lower limit of quantification in plasma was 2.5 ng/mL. This validated assay was successfully applied to a kinetic study of BAPN in mouse plasma and demonstrates that BAPN reaches the tumoral tissue.

Development of a sensitive method for the quantification of urinary 3-hydroxybenzo[a]pyrene by solid phase extraction, dansyl chloride derivatization and liquid chromatography-tandem mass spectrometry detection

A sensitive method for the quantification of urinary 3-OHBaP was developed by SPE, dansyl chloride derivatization and LC-MS/MS detection.

Development and in-house validation of a method for quantification of BMAA in mussels using dansyl chloride derivatization and ultra performance liquid chromatography tandem mass spectrometry

A new approach for the detection and quantification of β-N-methylamino-L-alanine (BMAA) in mussels using chemical derivatization with dansyl chloride and ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) is presented. The method, using dansyl chloride as the reagent, is simple, robust and cost efficient. Comparing the fragmentation patterns for derivatized BMAA and its isomer L-2,4-diaminobutyric acid derivatized a selective fragment for the derivatized BMAA was formed m/z 585 > m/z 71. To ensure an actual detection of BMAA, the ion ratio of the daughter ions m/z 71 and m/z 277 was calculated and compared with the calculated mean ion ratio. The method development resulted in a simplified sample preparation excluding solid phase extraction and, instead, performing filtration and dilution of the samples before the derivatization. Validation was performed and the limit of quantification was determined to be 0.15 μg g−1 wet mussel homogenate (33 fmol per injection) and the limit of detection was estimated to be 16 ng g−1 (4 fmol per injection). The intra- and inter-day precisions were within the accepted criteria and the recovery was about 83%. The stability of BMAA in the stock solution was at least 3 months and the stability of derivatized extracts in vials for the quantification was good for 27 days. This is the first quantification method for BMAA in mussels with extensive validation data published and the method was also applied on real mussel samples collected on the west coast of Sweden. The concentrations of BMAA in the mussel samples were determined to be between 0.27 and 1.6 μg g−1 wet mussel homogenates.

A sensitive and selective quantification of catecholamine neurotransmitters in rat microdialysates by pre-column dansyl chloride derivatization using liquid chromatography–tandem mass spectrometry

A rapid and sensitive liquid chromatography tandem mass spectrometry method for simultaneous quantification of catecholamine neurotransmitters in microdialysates was developed. The catecholamine neurotransmitters dopamine (DA) and norepinephrine (NE) were pre-column derivatized with dansyl chloride and analyzed. A gradient elution method was used to separate the analytes from the interferences on an Agilent Poroshell 120 EC-C18 outer porous micro particulate column. The method was robust and sensitive to determine with the lower limit of quantification value of 0.068pmol/mL and 0.059pmol/mL for DA and NE, respectively. It has acceptable precision and accuracy for concentrations over the standard curve range. The method was successfully applied for simultaneous quantitation of DA and NE in the prefrontal cortex (PFC) dialysates of rats obtained from a microdialysis study dosed with vehicle and atomoxetine through intra peritoneal (i.p.) route at a dose of 3mg/kg to monitor the change in extracellular concentrations. Thus, accomplishment of this method would facilitate the neurochemical monitoring for discovery of new chemical entities targeted for the treatment of attention deficit hyperactivity disorder (ADHD).

Enhanced screening efficiency for endocrine-disrupting chemicals in milk and powdered milk using UPLC/QTOF-MS by the introduction of dansyl chloride derivatisation

This study developed and validated a sensitive analytical method for simultaneous screening of four classes of endocrine-disrupting chemicals (i.e. progestogens, androgens, oestrogens and phenols) in milk and powdered milk using ultra-performance liquid chromatography (UPLC) coupled with electrospray ionisation quadrupole time-of-flight mass spectrometry (QTOF-MS). Dansylation of oestrogens and phenols enhanced the ionisation efficiency and shifted the ionisation mode from negative to positive, which allowed for the simultaneous analysis of four EDCs in one chromatographic run. An efficient sample pre-treatment minimised the matrix effects. The mass errors for the precursor and product ions for 26 target compounds varied between −2.8 and 2.3 mDa; and the limits of detection (signal-to-noise ratio = 3) for milk and powdered milk were less than 0.04 µg l−1 and 0.10 µg kg−1, respectively. The proposed method was successfully used to analyse multiple types of real samples, including normal temperature whole milk, infant formula and whole powdered milk. In 11 samples, two target compounds, progesterone and androstenedione, were detected. The progesterone concentrations ranged from 8.1 to 12.7 µg l−1 in milk, and from 1.2 to 32.0 µg kg−1 in infant formulas and whole powdered milks. The androstenedione concentrations varied from 0.39 to 0.79 µg l−1 in milks, and from 0.29 to 1.2 µg kg−1 in infant formulas and whole powdered milks. Two post-target compounds, one isomer of oestriol and 5α-dihydroprogesterone, were tentatively identified by post-target analysis in two of 11 real samples.

Quantitative analysis of estrogens and estrogen metabolites in endogenous MCF-7 breast cancer cells by liquid chromatography–tandem mass spectrometry

To study the roles of estrogens and estrogen metabolites (EMs) in breast carcinogenesis, we reported a quantitative liquid chromatography–tandem mass spectrometry (LC–MS/MS) method utilizing selective reaction mode (SRM) to analyze estrogens and EMs in the extracellular and intracellular compartments of endogenous MCF-7 breast cancer cells through simple ethyl acetate (EA) extraction and dansyl chloride derivatization. Under a 35-min LC gradient elution on a reversed phase C18 column, the method was shown to simultaneously quantify 12 estrogens and EMs: estrone (E1) and its 2-, 4-, 16α-hydroxy derivatives (2-OHE1, 4-OHE1, 16α-OHE1), and 2-, 4-methoxy derivatives (2-MeOE1, 4-MeOE1); 17β-estradiol (E2) and its 2-, 4-hydroxy derivative (2-OHE2, 4-OHE2) and 2- and 4-methoxy derivatives (2-MeOE2 and 4-MeOE2); and estriol (E3), using ethinylestradiol (EE2) as the internal standard (IS). Using a calibration curve–standard addition hybrid method, we were able to determine the amount of estrogens and EMs in not only the treated cells but also the non-treated cells. The limits of quantification (LOQs) were determined to range from 0.05–80 pg on column with an inter-batch accuracy around 72–123% and precision around 1–10%. Results indicated that trace amounts (<0.9 fg/cell) of E1 and E2 were present in both the extra- and intra-cellular compartments under non-treated condition but DMSO could induce E1 and E2 as well as trace amounts (<2.25 fg/cell) of EMs in the cell. E2 treatment substantially increased not only E1 and E2 in the intra-cellular (60 fg/cell) and extra-cellular (3000 fg/cell) compartment but also substantially induced EMs primarily in the extracellular compartment (0.6–25 fg/cell). These data implied that EMs could be quickly generated and distributed to the extracellular compartment by E2 within 24 h of treatment and DMSO solvent could potentially induce slight estrogen effects.

A new method for the determination of biogenic amines in cheese by LC with evaporative light scattering detector

This paper presents a new LC method with evaporative light scattering detection (ELSD), for the separation and determination of the biogenic amines (histamine, spermidine, spermine, tyramine, putrescine and β-phenylethylamine) which are commonly present in cheese, as their presence and relative amounts give useful information about freshness, level of maturing, quality of storage and cheese authentication. The LC–ELSD method is validated by comparison of the results with those obtained through LC–UV determination, based on a pre-column dansyl chloride derivatisation step. The obtained data demonstrate that both methods can be interchangeably used for biogenic amines determination in cheese. The new LC–ELSD method shows good precision and permits to achieve, for standard solutions, limit of detection (LOD) values ranging from 1.4 to 3.6mgL−1 and limit of quantitation (LOQ) values ranging from 3.6 to 9.3mgL−1. The whole methodology, comprehensive of the homogenization–extraction process and LC–ELSD analysis, has been applied in the analysis of a typical Calabria (Southern Italy) POD cheese, known as Caciocavallo Silano. The most aboundant amine found was histamine, followed, in decreasing order, by tyramine, spermine, putrescine, β-phenylethylamine and spermidine, for a total amount of 127mgkg−1. This value does not represent a possible risk for consumer health, according to the toxicity levels reported in literature and regarded as acceptable.

Quantification of piperazine phosphate in human plasma by high-performance liquid chromatography–electrospray ionization tandem mass spectrometry employing precolumn derivatization with dansyl chloride

This paper describes a novel method that combines dansyl chloride (DNS-CL) derivatization with high-performance liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI/MS/MS) for the sensitive and selective determination of piperazine phosphate in human plasma. After addition of ondansetron hydrochloride as internal standard (IS), piperazine phosphate was derivatized and then extracted with ethyl acetate. After being evaporated and reconstituted, the sample was analyzed using LC–ESI/MS/MS. Separation was achieved using an Agilent ZORBAX SB-C 18 (150 mm × 2.1 mm I.D., 3.5 μm) column and isocratic elution with 10 mM ammonium acetate solution (pH 3.0)–methanol (50: 50, v/v). Detection was performed on a triple-quadrupole mass spectrometer utilizing electrospray ionization (ESI) interface operating in positive ion and selected reaction monitoring (SRM) mode with the precursor to product ion transitions m/ z 320 → 171 for DNS-CL–piperazine phosphate and m/ z 294 → 170 for the IS. The method was fully validated for its selectivity, sensitivity, linearity, precision, accuracy, recovery, matrix effect and stability. The coefficient ( r) of piperazine phosphate with a linear range of 0.1–15 μg mL −1 was 0.9974–0.9995. The limit of detection and lower limit of quantification in human plasma were 0.01 and 0.1 μg mL −1, respectively. The validated LC–ESI/MS/MS method has been successfully applied to a bioequivalence study of piperazine phosphate trochiscus in Chinese healthy male volunteers.

Determination of 4-dimethylaminophenol concentrations in dog blood using LC–ESI/MS/MS combined with precolumn derivatization

A sensitive and reproducible LC–ESI/MS/MS method, which was combined with the precolumn dansyl chloride derivatization to enhance the signal intensity of analytes, was developed to determine blood 4-dimethylaminophenol (DMAP) concentrations. The linearity of the method was observed within the concentration range of 2–2000 ng/mL. The precision, accuracy, stability, recovery and matrix effect of the method were also investigated and found to meet the requirements for pharmacokinetic studies of the drug. By using this method, pharmacokinetic studies were conducted in dogs after i.m. and i.v. administrations. The results showed that DMAP could not only be absorbed into blood quickly after i.m., but also can be eliminated rapidly. Both i.m. and i.v. routes are appropriate for DMAP to be used in field first-aid. It has been proved that this LC–MS/MS combined with precolumn derivatization method can be used as a routine analytical method to provide enhanced measurements for blood DMAP concentrations. It is also useful for DMAP pharmacokinetic evaluation.

Trace analysis of icariin in human serum with dansyl chloride derivatization after oral administration of Epimedium decoction by liquid chromatography tandem mass spectrometry

Epimedium herbs are a type of complex traditional Chinese medicine (TCM) with high estrogenic bioactivity. The Epimedium herbal decoction mixture contains many compounds including icariin that can exert potent effects on numerous physiological processes related to human health. An ultrasensitive liquid chromatography tandem mass spectrometric (LC–MS/MS) method has been developed to determine trace levels of icariin in human serum with dansyl chloride derivatization after oral administration of the Epimedium herbal decoctions. The dansyl-icariin showed an intense protonated molecular ion at m/ z 910. The collision-induced dissociation of this ion formed a distinctive product at m/ z 764, corresponding to a characteristic removal of a rhamnose sugar moiety of icariin. The selected reaction monitoring, based on the m/ z 910 → 764 transition, was highly specific and ultrasenstive for icariin in human serum samples. The lower limit of quantitation was 10 pg/mL icariin spiked into blank serum. The ranges of coefficients of variation for interday assays and intraday assays were 0–15.0% and 1.1–17.5%, respectively, for a wide linear range from 10 pg/mL to 4 ng/mL. This method was successfully applied to measure trace levels of icariin in a human serum after oral administration of Epimedium decoction within 48 h for the first time.

Chemically enhanced liquid chromatography/tandem mass spectrometry determination of glutamic acid in the diffusion medium of retinal cells

A rapid, reproducible and highly sensitive method, based on liquid chromatography mass spectrometry, was developed for the determination of the excitatory amino acid glutamic acid released in the diffusion medium of control, ischemic and mutant cells from retinas. Signal intensity of glutamic acid was enhanced by dansyl chloride derivatization giving rise to a detection limit in the order of pmol/mL. Further, in HPLC-ESI-MS detection an MS-friendly dansyl group to glutamic acid enhanced both ionization efficiency in the ESI source and collision-activated dissociation in the collision cell. The sample processing procedure included liquid-liquid extraction, derivatization with dansyl chloride and a final cation-exchange extraction to generate clean extracts for LC/MS/MS analysis. This approach has been validated as sensitive, linear (20-300 ng/mL), accurate and precise for the differential quantification of glutamic acid in the diffusion medium of retina cells. This is the first report of using chemical derivatization to enhance MS/MS detection of the glutamic acid released in the diffusion medium of wild-type and mutant retina cells, under ischemic conditions.

Analysis of steroid estrogens in water using liquid chromatography/tandem mass spectrometry with chemical derivatizations

Even in trace amounts, estrogens such as 17beta-estradiol (E2), estrone (E1), estriol (E3), and 17alpha-ethinyl estradiol (EE2) may have adverse effects on humans and the aquatic ecosystem. Therefore, it is essential to be able to measure trace amounts of steroid estrogens in water. To date, most instruments are not sensitive enough to detect these chemicals in small samples of water. Sensitivity, however, may be improved by using appropriate derivatization reagents to modify the structures of these estrogens so that their ionization efficiency is increased, making them more detectable by liquid chromatography/mass spectrometry (LC/MS). This study uses dansyl chloride, 2-fluoro-1-methylpyridinium p-toluenesulfonate (FMPTS), and pentafluorobenzyl bromide (PFBBr) as derivatization reagents to react with the phenolic estrogens to make them more detectable in water. We also test how environmental matrices (wastewater effluent, river water, and drinking water) influence the detectability of these estrogens. Both qualitative and semi-quantitative comparisons of these derivatization methods were made. We found that dansyl chloride derivatives created signal intensities one or two orders of magnitude greater than those normally found in underivatized estrogen standards. The signals derived by FMPTS were analyte-dependent, and the products derived from E1, E2, and EE2 produced 2.19 to 12.1 times the signal intensity of underivatized E1, E2, and EE2. The product derived from E3 produced weaker signals than that produced by underivatized E3. The PFBBr derivatives produced signals that were as much as 5.8 times those found in the underivatized estrogens. When these derivatization methods were applied to river water, drinking water and effluents from a sewage treatment plant (STP), the different matrices were found to significantly suppress the signals if we used electrospray ionization, though this influence became less significant if we used atmospheric pressure chemical ionization. This study suggests that PFBBr derivatization can best be used for the detection of these estrogens in complex environmental matrices such as river water and STP effluents and that the dansyl chloride derivatization is best used for clean samples such as drinking water.

Fluorescent method for detection of cleaved collagens using O-phthaldialdehyde (OPA)

Analysis of collagen degradation remains an important but cumbersome task. Traditional methods with dansyl chloride derivatization of collagen have been used to quantify collagen damage. Fluorescent labeling reagents have been developed that offer advantages such as greater solubility in water and low background emission. One such reagent is o-phthalaldehyde (OPA). In this study, we used OPA as a means of detecting small amounts of degraded collagen. Collagen samples isolated from skin or heart were used for OPA conjugation to exposed amino termini (“opalation”). Experiments utilizing small samples aliquoted in microtiter plates were performed to evaluate effects of increasing concentrations of OPA, varying concentrations of collagen, and effects of matrix metalloproteinase (MMP) digestion. Results indicate that within 10 min of reaction, OPA can be used to detect relative differences in cleaved vs. uncleaved collagen from skin or heart. Heart samples obtained from regions of high MMP activity correlated with increased OPA fluorescence relative to tissue with lower MMP activity. On the basis of these results, we conclude that OPA has valuable practical advantages for analytical use in detecting cleaved collagen in small tissue samples.

Pharmacokinetics of eugenol in rainbow trout ( Oncorhynchus mykiss)

Clove oil, eugenol being its main constituent, has been used to anaesthetise fish for the last decade however no pharmacokinetic study of this drug has yet been realised in fish. The objective of the present study was to determine the pharmacokinetic parameters following immersion of rainbow trout ( Oncorhynchus mykiss) in a eugenol solution. A total of 30 fish ( n = 3 per time point) of similar length and weight were immersed in a solution of 75 mg/L of eugenol for 15 min. After thorough rinsing, individuals were placed in a recuperation tank in oxygenated conditioned water. Blood samples were collected at selected time points (at predose and at 0.25, 0.5, 1, 2, 4, 6, 8, 24 and 48 h) post-exposition. The samples were then submitted to mass spectrometry analysis using dansyl-chloride derivatisation. Pharmacokinetic results obtained are a C max of 10.53 μg/mL, a AUC 0− t 16.55 μg h/mL, AUC 0 − inf 17.04 μg h/mL and a half life of 12.14 h. These results indicate that eugenol is well absorbed and eliminated using a bath immersion at 4 °C. The long half life suggests that eugenol may accumulate in trout following repeated administration and further studies are required to evaluate its potential toxicity.

High performance liquid chromatography/tandem mass spectrometry determination of biogenic amines in typical Piedmont cheeses

The paper presents a new HPLC method, hyphenated with mass spectrometry detection, for the separation and determination of the biogenic amines that are most commonly present in cheese, namely cadaverine, histamine, spermidine, spermine, tyramine and tryptamine. The HPLC–MS/MS method is validated by comparison of the results with those obtained through a literature HPLC–UV determination, based on a pre-column dansyl chloride derivatisation step. The intercalibration is based on the statistical t-test for multiple samples that allows to compare simultaneously the results obtained with the two methods for more analytes and to decide, at a prefixed confidence level, if the two methods are inter-changeable. The new HPLC–MS/MS method, employed in selected reaction monitoring (SRM) mode, permits to achieve for standard solutions limit of detection (LOD) values ranging from 1.7 to 22.5 μg L −1 and LOQ (limit of quantitation) values ranging from 5.6 to 68.2 μg L −1. In order to apply the method in the analysis of cheeses, LOD and LOQ values have also been evaluated in “ricotta” cheese, in order to take as possible into account the matrix interference. In these conditions LODs range between 5.1 and 35.0 μg L −1 and LOQs between 14.2 and 101.2 μg L −1. The whole methodology, comprehensive of the homogenization-extraction process and HPLC–MS/MS analysis, has been applied in the analysis of three typical Piedmont (North-West Italy) cheeses, known as Toma Piemontese, Raschera and Castelmagno.

Development of a LC‐ESI/MS/MS assay for the quantification of vanillin using a simple off‐line dansyl chloride derivatization reaction to enhance signal intensity

Vanillin is responsible for producing the familiar smell of vanilla. Vanillin has many similarities with other flavor phenolic compounds and could potentially show similar pharmacological activity. A previously published analytical method was adapted, developed and tested. Vanillin was extracted from rat plasma using protein precipitation with acetone. Prior to LC-ESI/MS/MS analysis, an aliquot of the supernatant was used to proceed to the derivatization of vanillin and the internal standard with dansyl chloride to enhance signal intensity in positive electrospray mode. The chromatography was performed on a 100 x 2.1 mm C8 column and an isocratic mobile phase composed of 75:25 acetonitrile:0.5% formic acid in water with a flow rate fixed at 500 microL/min. A linear (weighted 1/concentration) relationship was used to perform the calibration over an analytical range of 10-10,000 ng/mL. The intra-batch precision and accuracy at the limit of quantitation (10 ng/mL), medium (500 ng/mL) and high (10,000 ng/mL) concentrations were 10.7, 7.0 and 7.2% and 103.5, 108.0 and 100.1%, respectively. The observed recovery was greater than 87% and no significant ionization suppression or matrix effect was observed. This LC-ESI/MS/MS method for the determination of vanillin in rat plasma provided results within generally accepted criteria used for bioanalytical assay.

Pharmacokinetics and anesthetic activity of eugenol in male Sprague–Dawley rats

Eugenol, the principle chemical constituent of clove oil, has recently been evaluated for its anesthetic and analgesic properties in fish and amphibians. The objective of this study was to determine the pharmacokinetic (PK) and anesthetic activity of eugenol in rats. Male Sprague-Dawley rats received single i.v. doses of eugenol (0, 5, 10, 20, 40 and 60 mg/kg) and anesthetic level was evaluated with the withdrawal reflex. For the 20 mg/kg dose level, blood and urinary samples were collected over 1 h for the PK assessment. Plasma and blood concentrations of eugenol, as well as metabolite identification in urine, were determined using a novel dansyl chloride derivatization method with liquid chromatography mass spectrometry (LC/MS/MS). PK parameters were calculated using noncompartmental methods. Eugenol-induced loss of consciousness in a dose-dependent manner, with mean (+/-SEM) recovery in reflex time of 167 +/- 42 sec observed at the highest dose level. Mean systemic clearance (Cl) in plasma and blood were 157 and 204 mL/min/kg, respectively. Glucuronide and sulfate conjugates were identified in urine. Overall, eugenol produced a reversible, dose-dependent anesthesia in male Sprague-Dawley rats.

The dynamics of biogenic amine formation in muscle tissue of carp (Cyprinus carpio)

The effect of various storage temperatures (3 &amp;plusmn; 2&amp;deg;C; 24 &amp;plusmn; 1&amp;deg;C and &amp;ndash;18 &amp;plusmn; 1&amp;deg;C) on the formation of biogenic amines in the muscle tissue of four hybrid lines of carp (Cyprinus carpio) with respect to microbial contamination was studied. Biogenic amines were determined by fluorescence detection HPLC based on pre-column dansylchloride derivatization. The studied microbiological parameters included total plate count, coliform bacteria count and psychrotrophic bacteria count. Concentrations of biogenic amines (putrescine, cadaverine, spermidine, spermine, tyramine, histamine and tryptamine) determined in the muscle tissue of hybrids of individual lines varied depending on temperature and storage period. All four lines showed a significant increase (P &amp;lt; 0.01) in putrescine, cadaverine, spermidine, spermine and tyramine concentrations over 7-day storage at 3 &amp;plusmn; 2&amp;deg;C (mean values 9.7 &amp;plusmn; 9.5, 3.4 &amp;plusmn; 4.2, 10.1 &amp;plusmn; 13.4, 6.3 &amp;plusmn; 2.1 and 26.2 &amp;plusmn; 4.7 mg/kg, respectively). No histamine was detected throughout the storage period. A health-threatening level of histamine was detected in samples stored at 24 &amp;plusmn; 1&amp;deg;C for 2 days, accompanied by marked sensory changes. The highest mean value was 333.0 &amp;plusmn; 100.0 mg/kg. Higher levels reaching significance (P &amp;lt; 0.01) compared with fresh fish were determined for putrescine (91.3 &amp;plusmn; 54.6 mg/kg), cadaverine (213.0 &amp;plusmn; 83.7 mg/kg), and tyramine (138.0 &amp;plusmn; 45.1 mg/kg). In samples stored at &amp;ndash;18 &amp;plusmn; 1&amp;deg;C for 3 months, the content of biogenic amines did not differ at the level of significance from the values determined in the fresh muscle tissue. Putrescine and cadaverine levels were the best correlates of growing bacteria count. Significant differences between individual hybrid lines of carp were detected at none of the storage temperatures, neither between individual biogenic amines nor between the microbiological indicators determined. &amp;nbsp; &amp;nbsp;

Determination of eugenol in rat plasma by liquid chromatography–quadrupole ion trap mass spectrometry using a simple off-line dansyl chloride derivatization reaction to enhance signal intensity

A rapid, selective and sensitive method was developed for the determination of eugenol concentration using an off-line dansyl chloride derivatization step to enhance signal intensity. The method consisted of a protein precipitation extraction followed by derivatization with dansyl chloride and analysis by full scan liquid chromatography electrospray quadrupole ion trap mass spectrometry (LC-ESI-QIT). The separation was achieved using a 100 x 2 mm C(8) analytical column combined with an isocratic mobile phase composed of 75:25 acetonitrile: 0.1% formic acid in water set at a flow rate of 0.25 mL/min. Signal intensity of the eugenol-dansyl chloride derivative was increased up to 100-fold as compared with the underivatized eugenol in positive electrospray mode. An analytical range of 100-20,000 ng/mL was used in the calibration curve of plasma and blood samples. The LOD observed was 0.5 pg injected on column. The novel method met all requirements of specificity, sensitivity, linearity, precision, accuracy and stability. In conclusion, a rapid and sensitive LC-ESI/MS/MS method using a derivatization agent was developed to enhance signal intensity of eugenol.