Simultaneous determination of 8-hydroxy-2′-deoxyguanosine and 5-methyl-2′-deoxycytidine in DNA sample by high performance liquid chromatography/positive electrospray ionization tandem mass spectrometry

8-Hydroxy-2′-deoxyguanosine (8-OHdG) and 5-methyl-2′-deoxycytidine (5-mdC) are utilized as useful biomarkers not only for early diagnosis but also for the detection and assessment of high-risk individuals. In the present study, a sensitive and specific method was developed for simultaneous determination of 8-OHdG and 5-mdC in DNA by high performance liquid chromatography/positive electrospray ionization tandem mass spectrometry. The limits of quantification for 8-OHdG and 5-mdC were 80 and 40 pg/ml, respectively. The calibration curves of 8-OHdG and 5-mdC were linear over the concentration range of 0.02–100 ng/ml and the correlation coefficients were higher than 0.9990. The intra-day and inter-day relative standard derivative values were in the range of 0.70–7.47% for 8-OHdG and 1.07–7.06% for 5-mdC, respectively. The recoveries were 93.4–108.5% for 8-OHdG and 87.4–104.9% for 5-mdC, respectively. This method was validated by determination of the background levels of 8-OHdG and 5-mdC in calf thymus DNA, and satisfactory results were obtained.     

Determination of lipoic acid in human plasma by HPLC-ECD using liquid–liquid and solid-phase extraction: Method development,validation and optimization of experimental parameters

A rapid, inexpensive, sensitive and specific HPLC-ECD method for the determination of lipoic acid in human plasma was developed and validated over the linearity range of 0.001–10 μg/ml using naproxen sodium as an internal standard (IS). Extraction of lipoic acid and IS from plasma (250 μl) was carried out with a simple one step liquid–liquid extraction using dichloromethane. Similarly solid-phase extraction was carried out using dichloromethane as extraction solvent. The separated organic layer was dried under the stream of nitrogen at 40 °C and the residue was reconstituted with the mobile phase. Complete separation of both lipoic acid and IS at 30 °C on Discovery HS C18 RP column (250 mm × 4.6 mm, 5 μm) was achieved in 6 min using 0.05 M phosphate buffer (pH 2.5 adjusted with phosphoric acid):acetonitrile (50:50, v/v) as a mobile phase pumped at the rate of 1.5 ml/min using electrochemical detector in DC mode at the detector potential of 1.0 V. The limit of detection and limit of quantification of lipoic acid were 200 pg/ml and 1 ng/ml, respectively. While on column limit of detection and limit of quantification of lipoic acid were 10 and 50 pg/ml, respectively. The absolute recoveries of lipoic acid with liquid–liquid and solid-phase extraction were 98.43, 95.65, 101.45, and 97.36, 102.73, 100.17% at 0.5, 1 and 5 μg/ml levels, respectively. Coefficient of variations for both intra-day and inter-day were between 0.28 and 4.97%. The method is validated and will be quite suitable for the analysis of lipoic acid in the plasma of human volunteers as well as patients with diabetes and cardiovascular diseases.     

Sensitive and rapid quantification of the cannabinoid receptor agonist naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-018) in human serum by liquid chromatography–tandem mass spectrometry

The current paper describes a validated method for the detection and quantification of naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-018), an ingredient of a herbal mixture called “Spice”, by means of HPLC–ESI–MS–MS in serum. Lower limit of detection and lower limit of quantification were 0.07 and 0.21 ng/ml, respectively. In 2 subjects who consumed ca. 50 μg/kg of JWH-018 by smoking, the active ingredient was detected by means of the described method. Thereby, the serum concentrations reached values of approx. 10 ng/ml and dropped within 3 h very fast (<10% of the measured maximum concentrations).     

Ultra sensitive quantitation of endogenous oxytocin in rat and human plasma using a two-dimensional liquid chromatography-tandem mass spectrometry assay

Oxytocin (OT) is a neuropeptide with an extremely low endogenous level (low pg/ml) in human plasma. It is very challenging to develop a highly sensitive assay to measure endogenous OT, including radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Electrospray ionization (ESI) liquid chromatography–tandem mass spectrometry (LC–MS/MS) can provide high-throughput and selective methods for quantification of peptides in biological samples. A novel and highly sensitive two-dimensional LC–MS/MS (2D-LC–MS/MS) assay combining solid-phase extraction (SPE) has been developed and validated for the determination of endogenous OT in both human and rat plasma. The lower limit of quantification (LLOQ) was 1.00 pg/ml for human and 50.0 pg/ml for rat. Human plasma diluted with water (1:6, v/v) was successfully optimized as a surrogate matrix for human to prepare standard curves without endogenous interference. The extraction efficiency and absolute recovery were above 65.8% using the HLB SPE procedure, and matrix effects were lower than 12%. The method was validated in the range of 1.00–250 pg/ml for human plasma and 50.0–10,000 pg/ml for rat plasma with precision less than 12.7% and accuracy less than 7%.     

Development and validation of a sensitive ultra performance liquid chromatography tandem mass spectrometry method for the analysis of fentanyl and its major metabolite norfentanyl in urine and whole blood in forensic context

Fentanyl and its major metabolite norfentanyl often occur in low doses in biological samples. Therefore, a highly sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed and fully validated. Sample preparation was performed on a mixed-mode cation exchange solid phase extraction (SPE) cartridge with an additional alkaline wash step to decrease matrix effects and thus increase sensitivity. Ionization of fentanyl and norfentanyl with electrospray ionization (ESI) was more efficient than atmospheric pressure chemical ionization (APCI). The use of a mobile phase of high pH resulted in higher ESI signals than the conventional low pH mobile phases. In the final method, gradient elution with 10 mM ammonium bicarbonate (pH 9) and methanol was performed. A comparison of columns with different internal diameter and/or smaller particles showed optimal resolution and sensitivity when an Acquity C18 column (1.7 μm, 2.1 mm × 50 mm) was used. Deuterium labeled internal standards were used, but with careful evaluation of their stability since loss of deuteriums was seen. With limits of detection of 0.25 pg/ml for fentanyl and 2.5 pg/ml for norfentanyl in urine and 5 pg/ml for fentanyl and norfentanyl in whole blood the presented method is highly appropriate for the analysis of fentanyl and norfentanyl in forensic urine and blood samples.     

Development of surface plasmon resonance imaging biosensors for detection of ubiquitin carboxyl-terminal hydrolase L1

We have developed a new method for highly selective determination of the ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) concentration using a surface plasmon resonance imaging (SPRI) technique and two different biosensors. UCH-L1 was captured from a solution by immobilized specific rabbit monoclonal antibody or specific LDN-57444 inhibitor due to formation of receptor–UCH-L1 complex on the biosensor surface. The analytically useful dynamic response range of both biosensors is between 0.1 and 2.5 ng/ml. The detection limit is 0.06 ng/ml for the biosensor with antibody and 0.08 ng/ml for the biosensor with inhibitor. Biosensors based on both antibody and inhibitor were found to be suitable for quantitative determination of the UCH-L1 and exhibit good tolerance to the potential interferents. Both biosensors gave comparable results in the range of 0 to 0.20 ng/ml for plasma samples and 0.30 to 0.49 ng/ml for cerebrospinal fluid samples. To validate the new methods, comparative determination of UCH-L1 by the commercial enzyme-linked immunosorbent assay (ELISA) kit was performed. In general, in terms of UCH-L1 concentration, a good correlation between SPRI and ELISA was found. The developed biosensors can be used successfully for the determination of UCH-L1 in body fluids.     

Development of enzyme-linked immunosorbent assay for determination of polybrominated diphenyl ether BDE-121

Our interests are in the development of immunoassay-based fast scanning methods for persistent organic pollutants. To develop the immunoassay method of polybrominated diphenyl ether (PBDE), a model compound of PBDE, 2,3′,4,5′,6-pentabromodiphenylether (BDE-121), has been chosen to develop its antibody and the competitive indirect enzyme-linked immunosorbent assay (ELISA) is developed. The hapten of BDE-121 containing reactive carboxylic acid was synthesized and conjugated to carrier proteins (bovine serum albumin [BSA] and ovalbumin [OVA]). Anti-BDE-121 polyclonal antibody was then developed in rabbits as a result of immunization with the BDE-121–BSA conjugate. The optimal amount of coating antigen BDE-121–OVA conjugate and the dilution of antiserum needed in the ELISA were determined with the checkerboard method, and the effects of the properties of PBST (phosphate-buffered saline and Tween 20) buffer (pH and salt concentration) and chemical solvent (types and concentrations) on the ELISA were investigated to achieve a rapid robust assay with high sensitivity. Under the optimized conditions, the developed indirect ELISA shows a linear detection range from 1.74 to 84.1 ng/ml, with an IC₅₀ value of 8.07 ng/ml and a detection limit of 0.644 ng/ml. In total, 11 kinds of compounds were tested for calculating the cross-reactivity, which was less than 8% for nearly all of them. Real samples were analyzed by the proposed immunoassay and gas chromatography/mass spectrometry (GC/MS).     

Determination of 8-iso-prostaglandin F(2alpha) in exhaled breath condensate using combination of immunoseparation and LC-ESI-MS/MS

Rapid and precise method for the determination of 8-iso-prostaglandin F(2alpha), an essential marker of the oxidative stress, in exhaled breath condensate (EBC) was developed. The protocol consisted of stable isotope dilution, immunoseparation combined with selective and sensitive LC-ESI-MS/MS operated in multiple reaction monitoring (MRM) mode. The imprecision of the developed method was below 8.8%, the parameter of mean inaccuracy was determined as <9.6% (0-250pg of 8-iso-prostaglandin F(2alpha)/ml EBC). The limit of detection (LOD) was 1 pg/ml EBC and limit of quantification (LOQ) 5 pg/ml EBC. A significant difference in 8-iso-prostaglandin F(2alpha) content between the group of asbestosis patients and healthy volunteers was found.     

Group determination of 14-membered macrolide antibiotics and azithromycin using antibodies against common epitopes

Erythromycin (ERY), clarithromycin (CLA), roxithromycin (ROX), and azithromycin (AZI) are macrolide antibiotics widely used in livestock and human medicine. Therefore, they are frequently found as pollutants in environmental water. A method based on indirect competitive enzyme-linked immunosorbent assay (ELISA) for group determination of these macrolides in foodstuffs, human biofluids, and water was developed. Carboxymethyloxime of clarithromycin (CMO–CLA) was synthesized and conjugated to bovine serum albumin (BSA) and gelatin to prepare immunogen and coating antigen with advantageous presentation of target epitopes, l-cladinose and d-desosamine, common for these analytes. Antibodies generated in rabbits were capable of recognizing ERY, CLA, and ROX as a group (100–150%), and AZI (12%) and did not cross-react with ERY degradants, which lack antibiotic activity. Assay displayed sensitivity of determination of 14-membered macrolides (IC₅₀ = 0.13–0.2 ng/ml) and low limit of detection (LOD) that was achieved at 0.02 to 0.03 ng/ml. It allowed performing analysis of milk, muscle, eggs, bovine serum, water, human serum and urine, and avoiding matrix effect without special pretreatment using simple dilution with assay buffer. For 15-membered macrolide AZI, the corresponding characteristics were IC₅₀ = 1.6 ng/ml and LOD = 0.14 ng/ml. The recoveries of veterinary and human medicine macrolides from corresponding matrices were validated and found to be satisfactory.     

Cumulative exposure assessment for trace-level polycyclic aromatic hydrocarbons (PAHs) using human blood and plasma analysis

Humans experience chronic cumulative trace-level exposure to mixtures of volatile, semi-volatile, and non-volatile polycyclic aromatic hydrocarbons (PAHs) present in the environment as by-products of combustion processes. Certain PAHs are known or suspected human carcinogens and so we have developed methodology for measuring their circulating (blood borne) concentrations as a tool to assess internal dose and health risk. We use liquid/liquid extraction and gas chromatography–mass spectrometry and present analytical parameters including dynamic range (0–250 ng/ml), linearity (>0.99 for all compounds), and instrument sensitivity (range 2–22 pg/ml) for a series of 22 PAHs representing 2–6-rings. The method is shown to be sufficiently sensitive for estimating PAHs baseline levels (typical median range from 1 to 1000 pg/ml) in groups of normal control subjects using 1-ml aliquots of human plasma but we note that some individuals have very low background concentrations for 5- and 6-ring compounds that fall below robust quantitation levels.     

A heterogeneous biotin–streptavidin-amplified enzyme-linked immunosorbent assay for detecting tris(2,3-dibromopropyl) isocyanurate in natural samples

Tris(2,3-dibromopropyl) isocyanurate (TBC) is a novel brominated flame retardant (BFR) that is widely used to substitute the prohibited BFRs throughout the world. With the development of research, the potential environmental and ecological harms of TBC have been revealed. For sensitive and selective detecting TBC, an indirect competitive biotin–streptavidin-amplified enzyme-linked immunosorbent assay (BA–ELISA) has been established in this study. The small molecular TBC–hapten was synthesized first; it mimicked the chemical structure of TBC and possessed a secondary amine group. The as-obtained hapten was then conjugated with carrier proteins to prepare artificial antigen. After immunization, the anti-TBC polyclonal antibody was obtained from separating rabbit serum. The procedures of this BA–ELISA were optimized. Under the optimal conditions, the limit of detection (IC₁₀) was 0.0067 ng/ml and the median inhibitory concentration (IC₅₀) was 0.66 ng/ml. Cross-reactivity values of the BA–ELISA with the tested TBC analogues were ?5%. This immunoassay was successfully applied to determine the TBC residue in river water samples that were collected near a BFR manufacturing plant. Satisfactory recoveries (92.1–109.2%) were obtained. The results indicated that this proposed BA–ELISA is suitable for the rapid and sensitive determining of TBC in environmental monitoring.     

Fasciola gigantica: Immunodiagnosis of fasciolosis by detection of circulating 28.5 kDa tegumental antigen

A monoclonal antibody (MoAb)-based sandwich ELISA was developed for the detection of circulating 28.5 kDa tegumental antigen (28.5 kDa TA) in the sera from mice experimentally infected with Fasciola gigantica. The MoAb was immobilized on a microtiter plate, and the antigen in the serum was captured and detected with biotinylated polyclonal rabbit anti TA antibody. The test could detect 28.5 kDa in the extracts of tegument (TA), whole body (WB) and excretory-secretory (ES) fractions at the concentrations of these crude antigens as low as 600 pg/ml, 16 and 60 ng/ml, respectively. This sandwich ELISA assay could detect the infection from day 1 to 35 post infection and showed that circulating level of 28.5 kDa TA peaked at day 1 post infection. In contrast, the antibody detection by indirect ELISA could only demonstrate the antibody level from 35 days post infection. The reliability of the assay method was evaluated using sera from mice infected with F. gigantica or Schistosoma mansoni, and hamsters infected with Opisthorchis viverrini, as well as healthy mice and hamsters. The sandwich ELISA exhibited a sensitivity and specificity at 94.55% and 100%, respectively, and with a positive predictive value of 100%, a negative predictive value of 97.39%, false positive rate of 0%, false negative rate of 5.50% and an accuracy of 98.2%. Thus, this detection method exhibited high specificity and sensitivity as well as could be used for early diagnosis of fasciolosis by F. gigantica.     

Determination of 8-hydroxydeoxyguanosine by an immunoaffinity chromatography-monoclonal antibody-based ELISA

The postulated importance of oxidative damage to DNA in aging and age-related degenerative pathologies such as cancer has prompted efforts to develop sensitive quantitation methods. 8-Hydroxy-2′-deoxyguanosine (8-OHdG) is a widely used marker for oxidative damage to DNA. To develop an immunoassay for quantitation of 8-OHdG, two monoclonal antibodies have been developed and characterized by competitive enzyme-linked immunosorbent assay (ELISA). Antibody 1F7 has 50% inhibition at 5 pmol 8-OHdG and 1 × 105 pmol dG, while antibody IF11 has 50% inhibition at 2.5 pmol 8-OHdG and 2000 pmol dG. Both antisera crossreact with guanosine and several structurally related derivatives, including 6-and 8-mercaptoguanosine, 8-bromoguanosine, 8-methylguanine, and 7-methylguanosine. Immunoaffinity columns were prepared with antibody 1F7, which exhibits higher selectivity than 1F11, to isolate 8-OHdG from DNA hydrolyzates followed by ELISA quantitation with antibody 1F11. This method allows the analysis of approximately one 8-OHdG/105 dG using 100μg DNA. To validate the assay, DNA extracted from human placental tissues were assayed by both ELISA and HPLC with electrochemical detection. Values by both methods correlated well (r = 0.87, p < 0.001), but the levels determined by ELISA were approximately sixfold higher than those determined by HPLC. This may be due to oligonucleotides detected by the ELISA but not the HPLC method or crossreactivity with other damaged bases present in the immunoaffinity purified material. Placental samples from current smokers had significantly higher 8-OHdG by ELISA than those from nonsmokers (p < 0.05). The method of immunoaffinity purification combined with ELISA quantitation has sufficient sensitivity for detecting 8-OHdG in human DNA samples. Although absolute values are higher than those determined by HPLC, the method provides a good alternative to the HPLC-EC method for monitoring relative oxidative damage in molecular epidemiological studies.     

Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) as a marker of oxidative stress in rheumatoid arthritis and aging: effect of progressive resistance training

Urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG), as a measure of oxidative stress, was measured before and after 12 weeks of progressive resistance strength training in 8 healthy elderly (65–80 yr) and eight healthy young (22–30 yr) men and women, and in eight adults (25–65 yr) with rheumatoid arthritis (RA).Training subjects exercised at 80% of their one-repetition maximum and performed eight repetitions per set, three sets per session, on a twice-weekly basis. 8-OHdG was measured at baseline and follow-up (at least 24 hr after the last exercise session) in the RA and elderly subject groups, and at baseline only in young subjects.Baseline 8-OHdG levels were greater among subjects with RA compared to both healthy young (P < 0.001) and elderly (P < 0.05) subjects. There were no changes in 8-OHdG levels in either RA or elderly subjects as a result of the strength training intervention.These results suggest that subjects with RA have higher levels of oxidative stress than young and elderly healthy individuals. Furthermore, there is no change in oxidative stress, measured by urinary 8-OHdG, in elderly healthy individuals or in subjects with RA after a 12-week strength training intervention.     

Association of adiponectin (AdipoQ) and sulphonylurea receptor (ABCC8) gene polymorphisms with Type 2 Diabetes in North Indian population of Punjab

In Type 2 Diabetes (T2D), adiponectin (AdipoQ) and sulphonylurea receptor genes (ABCC8) are important targets for candidate gene association studies. The single nucleotide polymorphisms (SNPs) in these genes have been associated with features of the metabolic syndrome across various populations. The present case–control study undertaken in the population of Punjab, evaluates the association of + 45T>G polymorphism in AdipoQ gene; and Exon16-3C>T as well as Exon18C>T polymorphisms in ABCC8 gene with T2D. These SNPs were genotyped in 200 T2D cases and 200 non-diabetic healthy controls using the PCR-RFLP method. The frequency of the minor G-allele for AdipoQ+ 45(T>G) polymorphism was significantly higher in T2D cases (29.0%) than in controls (21.5%) [P = 0.02, OR = 1.49 (1.07–2.04)]. The genetic model analysis revealed that the G-allele cumulatively provides nearly 1.59–1.78 fold increased risk to T2D under the additive (P = 0.009; OR = 1.59, 1.12–2.25 at 95% CI), dominant (TG/GG vs. TT) (P = 0.034, OR = 1.64, 1.04–2.56 at 95% CI) and codominant model (TG vs. TT/GG) (P = 0.014; OR = 1.78, 1.12–2.82 at 95% CI) after adjusting for confounding factors. However, no difference in the distribution of genotype and allele frequencies was observed for both the ABCC8 polymorphisms. The distribution of obesity profiles (BMI, WC and WHR) was also significantly different between cases and controls (P < 0.05). Higher BMI and central obesity were observed to increase the risk of T2D. G-allele of + 45(T>G) polymorphism in the adiponectin gene appears to be associated with increased risk of T2D, but the polymorphisms in sulphonylurea receptor gene do not seem to be associated with T2D in the population of Punjab.     

Capillary-based enzyme-linked immunosorbent assay for highly sensitive detection of thrombin-cleaved osteopontin in plasma

In this study, a highly sensitive capillary-based enzyme-linked immunosorbent assay (ELISA) has been developed for the analysis of picomolar levels of thrombin-cleaved osteopontin (trOPN), a potential biomarker for ischemic stroke, in human plasma. Using a square capillary coated with 8.5 μg/ml anti-human trOPN capture antibody for ELISA, the linear range obtained was 2 to 16 pM trOPN antigen. This concentration range was in the detection window of trOPN antigen in plasma samples. Compared with the conventional microplate-based ELISA, the current capillary technique significantly reduced the amounts of reagent from milliliter to microliter, reduced the analysis time from 8 to 3 h, and had a better sensitivity and detection limit performance from approximately 50 pM down to 2 pM of trOPN antigen. These results indicate that this capillary-based immunoassay is a potential tool for biomarker detection and may be useful in clinical trials and medical diagnostic applications.     

Application of one-step liquid chromatography–electrospray tandem MS/MS and collision-induced dissociation to quantification of ezetimibe and identification of its glucuronated metabolite in human serum: A pharmacokinetic study

A new one-step liquid chromatography–electrospray tandem MS/MS method is described to quantify ezetimibe (EZM) a novel lipid lowering drug in human serum. Also using collision-induced dissociation (CID) of the analyte, identification and chromatographic separation of its major metabolite, ezetimibe glucuronide (EZM-G) is achieved in this study. A thawed serum aliquot of 100 μL was deproteinated by addition of 500 μL methanol containing omeprazole as internal standard (I.S.). Separation of the drug, its metabolite and the I.S. were achieved using acetonitrile–water (70:30, v/v) as mobile phase at flow rate of 0.5 mL/min on a MZ PerfectSil target C18 column. Multiple reaction monitoring (MRM) mode of precursor–product ion transition (408.7 → 272.0 for EZM and 345 → 194.5 for the I.S.) was applied for detection and quantification of the drug while, EZM-G was chromatographically separated and identified using CID. The analytical method was linear over the concentration range of 1–32 ng/mL of EZM in human serum with a limit of quantification of 1 ng/mL. The coefficient variation values of both inter- and intra-day analysis were less than 8% whereas the percentage error was less than 3.7. The validated method was applied in a randomized cross-over bioequivalence study of two different EZM preparations in 24 healthy volunteers.     

Sandwich enzyme-linked immunosorbent assay for the quantification of human serum albumin fragment 408–423 in bodily fluids

Urinary levels of human serum albumin (hSA) fragment 408–423 have been proposed to represent an early marker for graft-versus-host disease (GvHD) and chronic kidney diseases. Here, we developed an enzyme-linked immunosorbent assay (ELISA) for the quantification of hSA(408–423). The sandwich ELISA has a detection limit of 0.5 ng/ml and is highly specific for hSA(408–423) because it does not cross-react with other albumin fragments or the full-length precursor. This ELISA allows rapid and convenient quantification of hSA(408–423) in bodily fluids, further clarifying the prognostic and diagnostic value of this peptide in GvHD, kidney disease, and other disorders.     

Development and validation of a nylon6 nanofibers mat-based SPE coupled with HPLC method for the determination of docetaxel in rabbit plasma and its application to the relative bioavailability study

A simple and sensitive HPLC method was established and validated for the determination of docetaxel (DTX) in rabbit plasma. Biosamples were spiked with paclitaxel (PCX) as an internal standard (I.S.) and pre-treated by solid-phase extraction (SPE). The SPE procedure followed a simple protein digestion was based on nylon6 electrospun nanofibers mats as sorbents. Under optimized conditions, target analytes in 500 μL of plasma sample can be completely extracted by only 2.5 mg nylon6 nanofibers mat and eluted by 100 μL solvent. The HPLC separation was obtained on C18 column and UV detector was used to quantify the target analytes. The extraction recovery was more than 85%; the standard curve was linear over the validated concentrations range of 10–5000 ng/mL and the limit of detection was 2 ng/mL. The inter-day coefficient of variation (CV%) of the calibration standards was below 5.0% and the mean accuracy was in the range of 92.8–113.4%. Moreover, analysing quality control plasma samples in 3 days, the results showed that the method was precise and accurate, for the intra- and inter-day CV% within 10% and the accuracy from 96.0% to 114.0%. The developed and validated method was successfully applied to relative bioavailability study for the preclinical evaluation of a new injectable DTX–sulfobutyl ether beta-cyclodextrin (DTX–SBE-β-CD) inclusion complex freeze-dried powder (test preparation), compared with the reference preparation (DTX injection, Taxotere^®) in healthy rabbits. On the basis of the mean AUC(0–t) and AUC(0–infinity), the relative bioavailability of the test preparation was found to be 113.1%.