Examination of the traction effect in an artificial muscle-type dynamic traction orthosis using computed tomography.

BACKGROUND: Dart-throwing motion from radial dorsiflexion to palmar flexion has recently attracted attention as a functional movement direction of the wrist joint. We developed a novel artificial muscle-type dynamic traction orthosis (DTSaM) that includes these movements. This prospective crossover controlled study aimed to compare the traction effects in the presence and absence of DTSaM using computed tomography. METHODS: Healthy participants with no history of finger disease (6 men: 6 fingers, 4 women: 4 fingers; average age [range]: 29.4 [34-24] years) were examined. The distance and area of the joint space between the radiolunate (RL) and capitolunate (CL) joints were evaluated using 2 types of computed tomography: automatic movement and DTSaM. RESULTS: Participants with DTSaM showed more dilated joint space distance on the dorsal and central sides ( p < 0.05) and larger joint space areas on the dorsal side ( p < 0.05) of the RL and CL joints than those without orthosis. Significant differences in the magnitude of change in each joint were observed between the RL and CL joints regarding the joint space distance on the dorsal ( p = 0.021) and central ( p = 0.038) sides and the joint space area on the dorsal side ( p = 0.044). CONCLUSIONS: The movement of the CL joint is important in the dorsiflexion direction. Our results suggest that the dilated CL joint allows dorsiflexion and that combined traction and dart-throwing motion exercises may be possible for wrist joint contracture.

On-line solid-phase extraction LC-MS/MS for the determination of Ac-SDKP peptide in human plasma from hemodialysis patients.

We developed a high-throughput method based on on-line solid-phase extraction liquid chromatography tandem mass spectrometry (SPE-LC-MS/MS) to determine N-terminal thymosin-ß fragment peptide (N-acetyl-seryl-aspartyl-lysyl-proline, Ac-SDKP) in human plasma samples. Quantification of Ac-SDKP was performed using direct injection for on-line SPE based on C(18), reversed-phase LC separation and stable isotope dilution electrospray ionization-MS/MS in multiple reaction-monitoring (MRM) mode. The Ac-SDKP-(13)C(6), (15)N(2) (m/z 496 → 137) was synthesized for the internal standard. The MRM ion for Ac-SDKP was m/z 488 → 129 (quantitative ion)/226. The limit of detection and lower limit of quantitation were 0.05 and 0.1 ng/mL in standard solution, respectively. Recovery values were 98.3-100.4% with inter-day (relative standard deviation, RSD, 0.4-14.1%) and intra-day (RSD, 0.8-19.7%) assays. This method was applied to the measurement of Ac-SDKP levels in plasma from hemodialyzed subjects. Concentrations were 0.59 ± 0.23 ng/mL (pre-hemodialyzed subjects, n = 9) and 0.44 ± 0.19 ng/mL (post-hemodialyzed subjects, n = 9). All plasma Ac-SDKP levels were decreased by dialysis. Thus, plasma Ac-SDKP was decreased through dialysis in chronic kidney disease. The findings in this study will be useful for the treatment of anemia in chronic kidney disease with dialysis.

Preparative purification of gentamicin components using high-speed counter-current chromatography coupled with electrospray mass spectrometry.

We developed a useful and preparative method based on high-speed counter-current chromatography with mass spectrometry (HSCCC/MS) to purify gentamicin C1a, C2/2a and C1 from standard powder. The analytes were purified on the HSCCC model CCC-1000 (multi-layer coil planet centrifuge) with a volatile two-phase solvent system composed of n-butanol/10% aqueous ammonia solution (50:50, v/v) and detected on an LCMS-2020EV quadrupole mass spectrometer fitted with an electrospray ionization (ESI) source system in positive ionization following scan mode (m/z 100-500). The HSCCC/ESI-MS peaks indicated that gentamicin C1a (m/z 450: [M+H](+)), C2/2a (m/z 464: [M+H](+)) and C1 (m/z 478: [M+H](+)) have the peak resolution values of 1.3 and 1.7 from 30 mg of loaded gentamicin powder. The HSCCC yielded 3.9 mg of gentamicin C1a, 12.6 mg of gentamicin C2/2a and 12.0 mg of gentamicin C1. These purified substances were analyzed by LC/MS with scan positive-mode. Based on the LC/MS chromatograms and spectra of the fractions, analytes were estimated to be over 95% pure. These gentamicin isomers of C1a, C2/2a and C1 were evaluated for their antibacterial activities. The overall results indicate that this approach of HSCCC/MS is a powerful technique for the purification of gentamicin components.

Screening assay of angiotensin-converting enzyme inhibitory activity from complex natural colourants and foods using high-throughput LC-MS/MS.

Inhibition of angiotensin-converting enzyme (ACE) by various foods decreases the blood pressure. ACE inhibitors derived from natural components may be of therapeutic value in preventive medicine. In this study, we report a novel screening assay of ACE inhibitors from complex natural colourants and foods that employ solid phase extraction (SPE), high-throughput liquid chromatography (LC) separation, and stable isotope dilution electrospray tandem mass spectrometry (SID-ESI-MS/MS). When a target sample was subjected to N-Hippuryl-His-Leu (HHL) and ACE in phosphate buffer (pH 7.4), generated hippuric acid (HA) was extracted by SPE. LC/SID-ESI-MS/MS detection of HA allowed us to accurately identify the effects of complex substances such natural colourants and foods that inhibit the ACE of HHL. The major HA and HA-d5 fragment ions at m/z 180→105 and 185→110 in the multiple reaction monitoring (MRM) mode can quantify levels that are lower than other methods. The LC/SID-ESI-MS/MS method described here is a rapid, selective, sensitive, and highly reproducible method for the determination of HA in various samples. Based on the assay developed, all samples such as natural colourants, infant formula, soy paste, ketchup, mayonnaise, wheat flour, orange juice, supplement drink, tea, and coffee could be accurately measured for ACE inhibition in various matrices. High-throughput LC/SID-ESI-MS/MS assay has no limitations in the evaluation of inhibition activity in various natural samples such as colour, high-matrix, and processed foods.

Analysis of a New Artificial Muscle Type Dynamic Orthosis for Wrist Joint Disease Using a Three-dimensional Motion Analyzer.

OBJECTIVES: Recently, the dart-throwing motion (DTM) has attracted attention as a functional direction of wrist joint motion. Consequently, we devised a new artificial muscle-type dynamic orthosis (Dynamic Traction Splint by Artificial Muscle, DTSaM) to reproduce DTM. This study analyzed the automatic motion of the wrist joint using a three-dimensional motion analysis system to assess how closely the DTSaM replicates DTM. METHODS: The DTSaM orthosis incorporates two McKibben-type rubber artificial muscles, and measurements were performed using image analysis software and a three-dimensional motion analysis system. The wrist radial angle (WRA) was defined as the angle between the line connecting the head of the index finger metacarpal to the radial styloid process and the line connecting the radial styloid process to the lateral epicondyle of the humerus. WRAs were investigated from 60° of palmar flexion to 60° of dorsiflexion. RESULTS: For dorsiflexion, comparisons of radial deviation and wrist ulnar angle (WUA) between the motion obtained using the DTSaM orthosis and active DTM showed a significant difference (P <0.05) at 30° and 60°, respectively. For palmar flexion, the same comparison showed a significant difference for both the ulnar and radial deviations (30°, P <0.05; and 60°, P <0.01, respectively). Furthermore, WUA showed a significant difference at 50° (P <0.05). Intraclass correlation coefficient analyses yielded good reliability with an average value of ≥0.8. CONCLUSION: The DTSaM orthosis produces a motion similar to the DTM. It is hoped that the use of the DTSaM orthosis will help to shorten the treatment period for patients with wrist disease.

Indices reflecting muscle contraction performance during exercise based on a combined electromyography and mechanomyography approach.

Electromyography (EMG) and mechanomyography (MMG) have been used to directly evaluate muscle function through the electromechanical aspect of muscle contraction. The purpose of this study was to establish new absolute indices to describe muscle contraction performance during dynamic exercise by combining EMG and displacement MMG (dMMG) measured simultaneously using our previously developed MMG/EMG hybrid transducer system. Study participants were eight healthy male non-athletes (controls) and eight male athletes. EMG and dMMG of the vastus medialis were measured for 30 s during four cycles of recumbent bicycle pedaling (30, 60, 90, and 120 W) and on passive joint movement. Total powers were calculated based on the time domain waveforms of both signals. Muscle contraction performance was verified with the slope of regression line (SRL) and the residual sum of squares (RSS) obtained from EMG and dMMG correlation. EMG and dMMG has increased with the work rate. Force and EMG were similar between groups, but dMMG showed a significant difference with load increase. Athletes had significantly higher SRL and significantly lower RSS than controls. The average value divided by SRL and RSS was higher in athletes than in controls. The indices presented by the combined approach of EMG and dMMG showed a clear contrast between the investigated groups and may be parameters that reflect muscle contraction performance during dynamic exercise.

Molecular Structure of Gardenia Blue Pigments by Reaction of Genipin with Benzylamine and Amino Acids.

Genipin was reacted with benzylamine and several amino acids to prepare gardenia blue (GB). The time-course of GB formation with benzylamine was monitored by high-performance liquid chromatography (HPLC), liquid chromatography time-of-flight mass spectrometry (LC-TOFMS), and 1H and 13C NMR measurements. In this experiment, we determined the molecular structures of some intermediates using accurate masses and additional NMR techniques such as heteronuclear multiple bond correlation (HMBC). GBs with amino acids (GB-AAs) were characterized by both liquid and solid-state NMR measurements. Interestingly, many significant peaks appeared in the solid-state NMR spectra, although the 13C NMR spectra from solution samples did not show any distinct peaks. Therefore, we determined that GB-AAs had an alternating copolymer structure composed of methyne and 5H-2-pyrindine, which was substituted by amino acids at N atom and linked with methyne at 5 and 7 positions. To confirm this molecular structure, the pyrolysis gas chromatography-mass spectrometry (GC-MS) measurement of GB-AAs was carried out, and 5H-2-pyrindine and its methyl derivatives were formed as main pyrolysis products from the polymer chains.

Determination of phthalates in diet and bedding for experimental animals using gas chromatography-mass spectrometry.

We have developed a gas chromatography-mass spectrometry method to measure five phthalates (dibutyl phthalate, butylbenzyl phthalate, di-2-ethylhexyl phthalate, diisooctyl phthalate, and diisononyl phthalate) in diets and beddings for experimental animals. The recoveries from diets and beddings spiked with five phthalates were 98.8%-148% with coefficients of variation of 0.4%-7.8% for diets and 94.7%-146% with coefficients of variation of 1.0%-5.0% for beddings. We analyzed commercial animal diets and beddings, and found that the levels of phthalates varied from sample to sample; the concentrations of five phthalates were 141-1,410 ng/g for diets and 20.5-7,560 ng/g for beddings.

Screening assay for metal-catalyzed oxidation inhibitors using liquid chromatography-mass spectrometry with an N-terminal beta-amyloid peptide.

Production of microregional catalytic reactive oxygen species (ROS) by metal-binding amyloid-beta (Abeta) peptides mediates the neurotoxicity of Alzheimer's disease, and inhibitors of this activity may be of therapeutic value. No current analytical methods target specific ROS inhibitors produced by metal-binding peptides. We report a screening assay for metal-catalyzed oxidation (MCO) inhibitors based on liquid chromatography-mass spectrometry (LC-MS) with a model N-terminal Abeta peptide (Abeta(1-6)). When subjected to MCO by Cu(II)/ascorbic acid, singly and doubly charged Abeta(1-6) molecules were observed at m/z 729.2 and 364.8 and m/z 685.3 and 343.3, respectively, corresponding to a decrease in mass of 45 and 89 Da compared with the model peptide. In contrast, H(2)O(2) did not modify the Abeta(1-6) peptide. Modified peptides were characterized by a specific MCO of Abeta(1-6), which contains both His and N-terminal Asp residues. LC-MS detection of the modified peptides allowed us to identify antioxidants that inhibit MCO of Abeta(1-6). MCO of the model peptide was inhibited by curcumin, but not dibutylhydroxytoluene, carotene, tocopherol, estradiol or nicotine, revealing a clear difference between curcumin and other antioxidants. This novel assay may allow for the identification of antioxidants that protect against MCO of peptides and proteins related to degenerative diseases.

LC-MS/MS and centrifugal ultrafiltration method for the determination of novobiocin in chicken, fish tissues, milk and human serum.

We present a rapid and simple method for detecting novobiocin in biologic samples using a methanol-based extraction of the tissue matrix and liquid chromatography with electrospray tandem mass spectrometry (LC-ESI-MS/MS) on positive mode. The sample, prepared using centrifugal ultrafiltration with 5.0% SDS, was directly injected into the LC-MS/MS. Chromatographic separation was performed on a TSK-GEL ODS 100 V column using 0.5% formic acid in water/methanol. The method was validated according to the Japanese Maximum Residue Limits recommendations. Detection was linear over a range of 5-100 ppb matrix solution (r>0.998). Novobiocin recovery values from chicken (0.05 ppm) and fish tissues (0.05 ppm), milk (0.08 ppm), and human serum (0.05 and 0.01 ppm) samples ranged from 71+/-1 to 95+/-2%.

Simultaneous determination of avermectins in bovine tissues by LC-MS/MS.

Analytical method for the simultaneous quantification of avermectins (AVMs), abamectin B1a, abamectin 8,9-Z isomer B1a, emamectine benzoate B1a, emamectine benzoate 8,9-Z isomer B1a, ivermectin, eprinomectin B1a, doramectin and moxidectin in bovine tissues (muscle, liver and fat) was developed by LC-MS/MS in electrospray positive ion mode. The separation was achieved on a short TSK-GEL ODS 100V column with the mobile phase consisting of acetonitrile and aquatic 0.1 mM ammonium formate containing 0.1% formic acid v/v at a flow rate of 0.2 mL/min with gradient elution. Liquid-liquid extraction with isooctane was used for the sample extraction/preparation of analytes in bovine samples. The linearity of the calibration curves was excellent in matrix-matched standards, and yielded the coefficients (r(2)=0.997-0.999, range from LOQ to 500, 1000 or 5000 ng/g) of determination of the target analytes. Recoveries were in the range of 87.9-99.8% with associated precision values (within-day: 1.5-7.4%, n=6, and between-day: 1.5-8.4% for 3 days) for repeatability and reproducibility. LC-MS/MS method has been proven to be highly efficient and suitable for the simultaneous determinations of eight AVMs in bovine tissue samples.

Supportive intervention using a mobile phone in behavior modification.

The authors previously developed a mobile ecological momentary assessment (EMA) system as a real-time data collection device using a mobile phone. In this study, a real-time advice function and real-time reporting function were added to the previous system as a supportive intervention. The improved system was found to work effectively and was applied to several clinical cases, including patients with depressive disorder, dizziness, smoking habit, and bronchial asthma. The average patient compliance rate was high (89%) without the real-time advice and higher (93%) with the advice. The trends in clinical data for patients using a mobile EMA with/without the new function were analyzed for up to several months. In the case of dizziness, an improving trend in its clinical data was observed after applying the real-time advice, and in the case of depressive disorder, a stabilizing trend was observed. The mobile EMA system with the real-time advice function could be useful as a supportive intervention in behavior modification and for motivating patients in self-management of their disease.

Rapid determination of carbamate pesticides in food using dual counter-current chromatography directly interfaced with mass spectrometry.

Dual counter-current chromatography (dual CCC)-tandem mass spectrometry (MS/MS) was successfully performed with a newly designed spiral column for dual CCC. The small column capacity required for directly coupling with electrospray MS/MS was accomplished by forming a rectangular spiral groove on a plastic disk and sealing it with a PTFE sheet. This novel dual CCC-MS/MS technique was successfully applied for the rapid determination of methomyl, fenobucarb and carbaryl pesticides in food. A two-phase solvent system of n-hexane-acetonitrile-0.1% formic acid (45:45:10) was suitable for both good dual CCC separation and sufficient ionization of pesticides. Recoveries of these three pesticides from mandarin orange and spinach samples fortified at 0.05 mg/kg were in the range of 93-107% with relative standard deviations of 2.4-3.8%.

Determination of avoparcin in animal tissues and milk using LC-ESI-MS/MS and tandem-SPE.

A highly sensitive and selective method using LC-ESI-MS/MS and tandem-SPE was developed to detect trace amounts of avoparcin (AV) antibiotics in animal tissues and milk. Data acquisition using MS/MS was achieved by applying multiple reaction monitoring of the product ions of [M + 3H](3+) and the major product ions of AV-alpha and -beta at m/z 637 --> 86/113/130 and m/z 649 --> 86/113/130 in ESI(+) mode. The calculated instrumental LODs were 3 ng/mL. The sample preparation was described that the extraction using 5% TFA and the tandem-SPE with an ion-exchange (SAX) and InertSep C18-A cartridge clean-up enable us to determine AV in samples. Ion suppression was decreased by concentration rates of each sample solution. These SPE concentration levels could be used to detect quantities of 5 ppb (milk), 10 ppb (beef), and 25 ppb (chicken muscle and liver). The matrix matching calibration graphs obtained for both AV-alpha (r >0.996) and -beta (r >0.998) from animal tissues and milk were linear over the calibration ranges. AV recovery from samples was higher than 73.3% and the RSD was less than 12.0% (n = 5).

[Analytical method for carbamate pesticides in processed foods by LC/MS/MS].

A rapid analytical method for the simultaneous determination of carbamate pesticides in processed foods was established by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). The pesticides were extracted from samples with acetonitrile using accelerated solvent extract equipment, except for the fine powder type spices, which were extracted in an ultrasonic bath. The crude extract was cleaned up with a multi-solvent GPC column (Shodex Asahipak GF-310 HQ) using acetonitrile as a mobile phase. The eluent from the column at the retention time between 13 to 18 min was concentrated under nitrogen gas and dissolved in a mixture of acetonitrile-water-0.2 mol/L ammonium formate buffer pH 6.0 (10 : 9 : 1). An aliquot was injected into the LC/MS/MS using electrospray ionization (ESI) with acquisition in the positive mode. The recoveries of 29 kinds of pesticide from dried fruits (raisin, prune and mango) and spices (turmeric, masala, sage, thyme and red pepper) fortified at levels of 0.1 and 0.01 microg/g were mostly in the range of 50 to 150% and those from soybean paste and soy sauce fortified at 0.01 microg/g were 46.9 to 122.6% (C.V. 3.8 to 37.6%), except for 4 kinds of pesticide. The determination limits (S/N> or =10) corresponded to 0.001 to 0.05 mug/g of the pesticides in red pepper.

A High-Fat and High-Cholesterol Diet Induces Cardiac Fibrosis, Vascular Endothelial, and Left Ventricular Diastolic Dysfunction in SHRSP5/Dmcr Rats.

AIM: Non-alcoholic steatohepatitis (NASH) increases cardiovascular risk regardless of risk factors in metabolic syndrome. However, the intermediary factors between NASH and vascular disease are still unknown because a suitable animal model has never been established. The stroke-prone (SP) spontaneously hypertensive rat, SHRSP5/Dmcr, simultaneously develops hypertension, acute arterial lipid deposits in mesenteric arteries, and NASH when feed with a high-fat and high-cholesterol (HFC) diet. We investigated whether SHRSP5/Dmcr affected with NASH aggravates the cardiac or vascular dysfunction. METHOD: Wister Kyoto and SHRSP5/Dmcr rats were divided into 4 groups of 5 rats each, and fed with a SP or HFC diet. After 8 weeks of HFC or SP diet feeding, glucose and insulin resistance, echocardiography, blood biochemistry, histopathological staining, and endothelial function in aorta were evaluated. RESULTS: We demonstrate that SHRSP5/Dmcr rats fed with a HFC diet presented with cardiac and vascular dysfunction caused by cardiac fibrosis, endothelial dysfunction, and left ventricular diastolic dysfunction, in association with NASH and hypertension. These cardiac and vascular dysfunctions were aggravated and not associated with the presence of hypertension, glucose metabolism disorder, and/or obesity. CONCLUSIONS: SHRSP5/Dmcr rats may be a suitable animal model for elucidating the organ interaction between NASH and cardiac or vascular dysfunction.

Muscle contraction accelerates IL-6 mRNA expression in the rat masseter muscle.

OBJECTIVE: This study was conducted to determine if interleukin-6 (IL-6) and interleukin-1beta (IL-1beta) mRNA expression increase in response to muscle contraction caused by repetitive electrical stimulation of the rat masseter muscle. METHODS: Male Wistar rats weighing 140-160 g were divided randomly into the following three groups: electrical stimulation (ES) group (n=21), carrageenan injection (CI) group (n=24), and ES under dantrolene sodium (muscle relaxant) injection (ESDI) group (n=7). ES or CI was done to the left masseter; and mock ES or mock CI to the right. Muscle tissues on both sides were sampled for total RNA isolation. Real-time RT-PCR was performed, with the cyclophilin A (CypA) mRNA level in each sample as an internal control. Mean relative IL-6 (il-6/cypA) and IL-1beta (il-1beta/cypA) mRNA levels were compared between the experimental and mock-treated sides within each group. RESULTS: Mean IL-6/CypA levels in the ES- or CI-treated muscle significantly increased, without any significant incremental change observed in either mock-treated muscle. Interestingly, the increase in the il-6/cypA level caused by the ES was suppressed by the injection of dantrolene sodium in the ESDI group. Furthermore, the mean il-1beta/cypA level in the CI-treated masseter also significantly increased without any significant incremental change observed in the mock-treated muscle. However, there was no significant difference in the mean il-1beta/cypA levels in the masseter between the ES- and the mock-treated sides. CONCLUSIONS: These results show that IL-6 mRNA expression in the rat masseter muscle was accelerated by the CI or by repetitive muscle contraction induced by ES. Since the mRNA level of IL-1beta, a well-known proinflammatory cytokine, was not altered by the contraction, the accelerated IL-6 mRNA expression elicited by the muscle contraction does not seem to be related to local inflammation.

Estimating the minimum stimulation frequency necessary to evoke tetanic progression based on muscle twitch parameters.

The summation of the muscle force caused by an increase in the firing rate is named a tetanic contraction (tetanus), and the minimum stimulation frequency necessary to evoke an unfused/fused tetanus is related to the contraction time (CT) and relaxation time (RT) of the twitch. In particular, the fusion index (FI) is a very useful indicator, and it is used to evaluate the change in the muscle fiber component ratio. However, the measurement of the FI is invasive, because most patients experience pain during the electrical stimulation for tetanus. We expect that the twitch parameters CT and RT can substitute for the FI in the future. We found that the minimum stimulation frequency necessary to evoke the unfused/fused tetanus can be estimated from the twitch parameters as a first step. The results showed that (1) the minimum stimulation frequencies calculated from twitch parameters during unfused/fused tetanus were very similar to those calculated from FI parameters, and (2) they were also strongly correlated with FI parameters regardless of fiber components. The basic characteristics of tetanic progression in different fiber types could be estimated from twitch parameters.

Effect of different surgical procedures on the accuracy of prediction of the plasma concentration of fentanyl: comparison between mastectomy and laparoscopic prostatectomy.

BACKGROUND: The accuracy of simulation-predicted fentanyl concentration in different types of surgical procedure is not fully understood. We wished to estimate the effect of different types of surgical procedure on the accuracy of such simulations. FINDINGS: Fifty patients who had undergone elective mastectomy or laparoscopic prostatectomy (American Society of Anesthesiologists physical status = I-II) were enrolled. Anesthesia was maintained throughout surgery with sevoflurane and a bolus infusion of fentanyl. A maintenance infusion was administered with 8 mL/kg/h Ringer's acetate solution from the start of anesthesia to completion of blood sampling. An infusion to compensate for blood loss was administered (one to two volumes of hydroxyethyl starch). A blood sample was drawn every 30 min during anesthesia.We measured the plasma concentration of fentanyl in 358 samples from 50 patients. The plasma concentration of fentanyl was correlated significantly with the simulated predicted fentanyl concentration (r = 0.734, P < 0.01) but 36.0% of all samples had a difference greater than ±0.5 ng/mL. Approximately 0.3 ng/mL of a fixed bias was shown throughout mastectomy. During laparoscopic prostatectomy, the fixed bias gradually became negative from ≈0.3 to -0.3 ng/mL as the sampling stage proceeded. CONCLUSIONS: The predicted concentration of fentanyl was significantly correlated with the plasma concentration of fentanyl (r = 0.734). However, there were different patterns of a fixed bias between mastectomy and laparoscopic prostatectomy groups. We should pay attention to this tendency among different surgical procedures. TRIAL REGISTRATION: UMIN000005110.

Application of dual counter-current chromatography for rapid sample preparation of N-methylcarbamate pesticides in vegetable oil and citrus fruit.

Dual counter-current chromatography (dual CCC) has been successfully applied to rapid sample preparation for the simultaneous determination of residual carbaryl, fenobucarb and methomyl in vegetable oil and citrus fruit. The citrus fruit samples were extracted with n-hexane solution containing stable isotopically labeled internal standards (methomyl-d3, fenobucarb-d3 and carbaryl-d9), and applied to dual CCC using a two-phase solvent system of n-hexane-acetonitrile to purify the carbamate pesticides from aliphatic sample matrix. The coiled column was rotated at 420 rpm, the lower mobile phase was introduced through the head toward the tail, and the upper mobile phase in the opposite direction. Due to the high partition efficiency of dual CCC, the lower phase fraction collected from 2 to 5 min after injection could be subjected to flow-injection tandem mass spectrometry directly after concentration. Repetitive sample injection can be performed at high reproducibility without a risk of contamination from the compounds retained in the column.