Association of AI-determined Kellgren-Lawrence grade with medial meniscus extrusion and cartilage thickness by AI-based 3D MRI analysis in early knee osteoarthritis.

The associations among Kellgren-Lawrence (KL) grade, medial meniscus extrusion (MME), and cartilage thickness in knee osteoarthritis (OA) remain insufficiently understood. Our aim was to determine these associations in early to moderate medial tibiofemoral knee OA. We included 469 subjects with no lateral OA from the Kanagawa Knee Study. KL grade was assessed using artificial intelligence (AI) software. The MME was measured by MRI, and the cartilage thickness was evaluated in 18 subregions of the medial femorotibial joint by another AI system. The median MME width was 1.4 mm in KL0, 1.5 mm in KL1, 2.4 mm in KL2, and 6.0 mm in KL3. Cartilage thinning in the medial femur occurred in the anterior central subregion in KL1, expanded inwardly in KL2, and further expanded in KL3. Cartilage thinning in the medial tibia occurred in the anterior and middle external subregions in KL1, expanded into the anterior and middle central subregions in KL2, and further expanded in KL3. The absolute correlation coefficient between MME width and cartilage thickness increased as the KL grade increased in some subregions. This study provides novel insights into the early stages of knee OA and potentially has implications for the development of early intervention strategies.

Morphological analysis of three-dimensional MR images of patellofemoral joints in asymptomatic subjects.

The existing methods for analyzing patellofemoral (PF) osteoarthritis (OA) are limited. Our purpose was to clarify the frequency, localization, and morphological progression of PFOA by observing three-dimensional (3D) magnetic resonance (MR) images from a cohort population. The subjects were 561 patients aged 30-79 years from the Kanagawa Knee Study who had not visited a hospital for more than three consecutive months for knee symptoms. MR images of the PF joints, separated into the medial and lateral types, were presented in order of the highest to lowest patella cartilage area ratios. Cartilage defects in the patella were detected in 37 subjects (6.6%). Medial lesions (4.6%) were significantly more frequent than lateral lesions (2.0%) (p < 0.01). For both medial and lateral lesions, the patellar cartilage defects were divided into confined and unconfined types. The 3D MR images of the PF joint showed that the patellar cartilage defect occurred along each ridge of the femoral trochlea. The 3D MR images revealed a 6.6% prevalence of patellar cartilage defects, higher in the medial than lateral regions. The 3D MR images can easily determine PF morphology and cartilage defect location, making them useful in understanding the pathophysiology and etiology of PFOA.

Medial Tibial Osteophyte Width Strongly Reflects Medial Meniscus Extrusion Distance and Medial Joint Space Width Moderately Reflects Cartilage Thickness in Knee Radiographs.

BACKGROUND: The presence of medial tibial osteophytes on knee radiographs suggests cartilage wear, but may be associated with medial meniscus extrusion (MME). The joint space width of the medial compartment consists anatomically of cartilage and the medial meniscus, but which is most responsible for joint space narrowing remains unclear. Magnetic resonance imaging (MRI) reveals MME and cartilage thickness. PURPOSES: To determine which radiographic medial tibial osteophyte width correlates better with cartilage thickness or MME distance and which radiographic medial joint space width correlates better with cartilage thickness or MME distance. STUDY TYPE: Cross-sectional. POPULATION: Total of 527 subjects, 253 females and 274 males, aged 30-79 years, included in the Kanagawa Knee Study. FIELD STRENGTH/SEQUENCE: 3 T/fat-suppressed spoiled gradient echo and proton density weighted. ASSESSMENT: The medial tibial osteophyte width and "the minimum joint space width at the medial compartment" (mJSW) were measured from plain radiographs. The cartilage region was automatically extracted from MRI data using software. The medial femoral and tibial cartilage regions were each divided into nine subregions, and the average thickness of the cartilage was determined in each region and subregion. MME was manually measured by two orthopedic surgeons using MRI coronal section images. STATISTICAL TESTS: Pearson's correlation coefficient and their comparison, with P < 0.05 considered statistically significant. RESULTS: The absolute values of the correlation coefficients were 0.33 at maximum between osteophyte width and cartilage thickness and 0.76 between osteophyte width and MME; the value was significantly higher with MME than with cartilage thickness (P < 0.001). The absolute values of the correlation coefficients were 0.50 at maximum between mJSW and cartilage thickness and 0.16 between mJSW and MME; the value was significantly higher with cartilage thickness than with MME (P < 0.001). DATA CONCLUSION: The medial tibial osteophyte width strongly reflected MME and the medial joint space width moderately reflected cartilage thickness. LEVEL OF EVIDENCE: 3 TECHNICAL EFFICACY STAGE: 3.

Difference in the joint space of the medial knee compartment between full extension and Rosenberg weight-bearing radiographs.

OBJECTIVES: Radiographs are the most widespread imaging tool for diagnosing osteoarthritis (OA) of the knee. Our purpose was to determine which of the two factors, medial meniscus extrusion (MME) or cartilage thickness, had a greater effect on the difference in the minimum joint space width (mJSW) at the medial compartment between the extension anteroposterior view (extension view) and the 45° flexion posteroanterior view (Rosenberg view). METHODS: The subjects were 546 participants (more than 50 females and 50 males in their 30 s, 40 s, 50 s, 60 s, and 70 s) in the Kanagawa Knee Study. The mJSW at the medial compartment was measured from both the extension and the Rosenberg views, and the "mJSW difference" was defined as the mJSW in the Rosenberg view subtracted from the mJSW in the extension view. The cartilage region was automatically extracted from MRI data and constructed in three dimensions. The medial region of the femorotibial joint cartilage was divided into 18 subregions, and the cartilage thickness in each subregion was determined. The MME was also measured from MRI data. RESULTS: The mJSW difference and cartilage thickness were significantly correlated at 4 subregions, with 0.248 as the highest absolute value of the correlation coefficient. The mJSW difference and MME were also significantly correlated, with a significantly higher correlation coefficient (0.547) than for the mJSW difference and cartilage thickness. CONCLUSIONS: The MME had a greater effect than cartilage thickness on the difference between the mJSW at the medial compartment in the extension view and in the Rosenberg view. KEY POINTS: • The difference in the width at the medial compartment of the knee between the extension and the flexion radiographic views was more affected by medial meniscus extrusion than by cartilage thickness.

Relationship between medial meniscus extrusion and cartilage measurements in the knee by fully automatic three-dimensional MRI analysis.

BACKGROUND: We developed a fully automatic three-dimensional knee MRI analysis software that can quantify meniscus extrusion and cartilage measurements, including the projected cartilage area ratio (PCAR), which represents the ratio of the subject's actual cartilage area to their ideal cartilage area. We also collected 3D MRI knee data from 561 volunteers (aged 30-79 years) from the "Kanagawa Knee Study." Our purposes were to verify the accuracy of the software for automatic cartilage and meniscus segmentation using knee MRI and to examine the relationship between medial meniscus extrusion measurements and cartilage measurements from Kanagawa Knee Study data. METHODS: We constructed a neural network for the software by randomly choosing 10 healthy volunteers and 103 patients with knee pain. We validated the algorithm by randomly selecting 108 of these 113 subjects for training, and determined Dice similarity coefficients from five other subjects. We constructed a neural network using all data (113 subjects) for training. Cartilage thickness, cartilage volume, and PCAR in the medial femoral, lateral femoral, medial tibial, and lateral tibial regions were quantified by using the trained software on Kanagawa Knee Study data and their relationship with subject height was investigated. We also quantified the medial meniscus coverage ratio (MMCR), defined as the ratio of the overlapping area between the medial meniscus area and the medial tibial cartilage area to the medial tibial cartilage area. Finally, we examined the relationship between MMCR and PCAR at middle central medial tibial (mcMT) subregion located in the center of nine subregions in the medial tibial cartilage. RESULTS: Dice similarity coefficients for cartilage and meniscus were both approximately 0.9. The femoral and tibial cartilage thickness and volume at each region correlated with height, but PCAR did not correlate with height in most settings. PCAR at the mcMT was significantly correlated with MMCR. CONCLUSIONS: Our software showed high segmentation accuracy for the knee cartilage and meniscus. PCAR was more useful than cartilage thickness or volume since it was less affected by height. Relations ips were observed between the medial tibial cartilage measurements and the medial meniscus extrusion measurements in our cross-sectional study. TRIAL REGISTRATION: UMIN, UMIN000032826 ; 1 September 2018.

Development of miniaturized hollow-fiber assisted liquid-phase microextraction with in situ acyl derivatization followed by GC-MS for the determination of benzophenones in human urine samples.

A simple and highly sensitive method that involves miniaturized hollow fiber assisted liquid-phase microextraction (HF-LPME) with in situ acyl derivatization and GC-MS was developed for the determination of benzophenone (BP) and related compounds in human urine samples. The limits of detection (S/N = 3) and quantification (S/N > 10) of BPs in human urine samples are 0.01 to 0.05 and 0.05 to 0.2 ng ml(-1), respectively. The average recoveries of BPs (n = 5) in human urine samples spiked with 10 and 50 ng ml(-1) BPs are 93.1 to 106.7% (RSD: 1.5 to 8.4%) and 96.3 to 101.5% (RSD: 3.0 to 7.7%), respectively. When the proposed method was applied to human urine samples, BPs were detected at the sub ng ml(-1) level.

Miniaturized hollow fiber assisted liquid-phase microextraction and gas chromatography-mass spectrometry for determination of benzophenone and derivates in human urine sample.

The determination of benzophenones (BPs) in human urine sample by miniaturized hollow fiber assisted liquid-phase microextraction (HF-LPME) and gas chromatography-mass spectrometry (GC-MS) is described. As analytes, BP, its metabolites benzhydrol (BP-OH) and 2-hydroxybenzophenone (2OH-BP), and its derivatives 2-hydroxy-4-methoxybenzophenone (BP-3) and 2-hydroxy-4-methoxy-4'-methylbenzophenone (BP-10) were selected. The detection limit and the quantification limit of BPs in human urine sample are 5-10 and 20-50 pg mL(-1), respectively. The calibration curve for BPs is linear with correlation coefficient higher than 0.99 in the range of 0.02-10 or 0.05-10 ng mL(-1). The average recoveries of BPs in human urine samples spiked with 0.5 and 5 ng mL(-1) BPs are 89.8-100.2% (RSD: 2.5-9.3%) and 89.3-99.9% (RSD: 2.9-3.7%), respectively. Ten human urine samples were analyzed using the present method. BP-OH and BP-3 were detected in all the samples within the range of 0.24-5.91 and 0.43-5.17 ng mL(-1), respectively. This simple, sensitive, and selective analytical method was successfully applied to the determination of trace amounts of BPs in human urine samples.

Stir bar sorptive extraction with in situ derivatization and thermal desorption-gas chromatography-mass spectrometry for trace analysis of methylmercury and mercury(II) in water sample.

A method for the trace analysis of methylmercury (MeHg) and Hg(II) in water sample was developed, which involved stir bar sorptive extraction (SBSE) with in situ alkylation with sodium tetraethylborate and thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS). The limits of quantification of MeHg and Hg(II) are 20 and 10 ng L(-1) (Hg), respectively. The method shows good linearity and the correlation coefficients are higher than 0.999. The average recoveries of MeHg and Hg(II) in tap or river water sample are 102.1-104.3% (R.S.D.: 7.0-8.9%) and 105.3-106.2% (R.S.D.: 7.4-8.5%), respectively. This simple, accurate, sensitive, and selective analytical method may be used in the determination of trace amounts of MeHg and Hg(II) in tap and river water samples.

Measurement of benzophenones in human urine samples by stir bar sorptive extraction and thermal desorption-gas chromatography-mass spectrometry.

Determination of benzophenones (BPs) in human urine samples by stir bar sorptive extraction (SBSE) and thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS) is described. As analytes, BP, its metabolites benzhydrol (BP-OH) and 2-hydroxybenzophenone (2OH-BP), and its derivatives 2-hydroxy-4-methoxybenzophenone (BP-3) and 2-hydroxy-4-methoxy-4'-methylbenzophenone (BP-10) were selected. After enzymatic hydrolysis, a polydimethylsiloxane (PDMS) stir bar was placed in a urine sample diluted 1:1 with water and stirred for 60 min at room temperature. The limit of quantification (LOQ) of BPs is 0.2-0.5 ng ml(-1) (ppb). The method showed linearity over the calibration range (0.2-10 or 0.5-10 ng ml(-1)), and the correlation coefficients were equal to or higher than 0.993 for all of the analytes. The average recoveries of BPs were equal to or higher than 98.7% (RSD: 1.5-4.8%, n = 6).

Determination of urinary triclosan by stir bar sorptive extraction and thermal desorption-gas chromatography-mass spectrometry.

We have developed an analytical method for the determination of urinary 5-chloro-2-(2,4-dichlorophenoxy)phenol (triclosan), which utilizes stir bar sorptive extraction (SBSE) and thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS). Human urine sample is de-conjugated by treatment with beta-glucuronidase and sulfatase. A stir bar coated with polydimethylsiloxane (PDMS) is added to the urine sample in a vial and the sample is stirred for 60 min at room temperature (25 degrees C). Then, the PDMS stir bar is subjected to TD-GC-MS. The detection limit of triclosan is 0.05 ng mL(-1). The method shows linearity over the calibration range (0.1-10 ng mL(-1)) and the correlation coefficient (r) is higher than 0.993 for triclosan standard solution. The average recoveries of triclosan in human urine sample are 102.8-113.1% (RSD: 2.4-6.7%). This simple, sensitive, and selective analytical method may be used in the determination of trace amounts of triclosan in human urine samples.

Mercury speciation and analysis in drinking water by stir bar sorptive extraction with in situ propyl derivatization and thermal desorption-gas chromatography-mass spectrometry.

A method for mercury analysis and speciation in drinking water was developed, which involved stir bar sorptive extraction (SBSE) with in situ propyl derivatization and thermal desorption (TD)-GC-MS. Ten millilitre of tap water or bottled water was used. After a stir bar, pH adjustment agent and derivatization reagent were added, SBSE was performed. Then, the stir bar was subjected to TD-GC-MS. The detection limits were 0.01 ng mL(-1) (ethylmercury; EtHg), 0.02 ng mL(-1) (methylmercury; MeHg), and 0.2 ng mL(-1) (Hg(II) and diethylmercury (DiEtHg)). The method showed good linearity and correlation coefficients. The average recoveries of mercury species (n=5) in water samples spiked with 0.5, 2.0, and 6.0 ng mL(-1) mercury species were 93.1-131.1% (RSD<11.5%), 90.1-106.4% (RSD<7.8%), and 94.2-109.6% (RSD<8.8%), respectively. The method enables the precise determination of standards and can be applied to the determination of mercury species in water samples.

Stir bar sorptive extraction and thermal desorption-gas chromatography-mass spectrometry for trace analysis of triclosan in water sample.

A simple and highly sensitive method called stir bar sorptive extraction (SBSE) and thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS), which is used for the determination of trace amounts of 5-chloro-2-(2,4-dichlorophenoxy)phenol (triclosan) in river water samples, is described. A stir bar coated with polydimethylsiloxane (PDMS) is added to a 10 mL water sample and stirring is carried out for 120 min at room temperature (25 degrees C) in a vial. Then, the PDMS stir bar is subjected to TD-GC-MS. The detection limit of triclosan is 5 ng L(-1) (ppt). The method shows linearity over the calibration range (0.02-20 microg L(-1)) and the correlation coefficient is higher than 0.997 for triclosan standard solution. The recovery of triclosan in river water samples ranges from 91.9 to 108.3% (RSD: 4.0-7.0%). This simple, accurate, sensitive, and selective analytical method may be used in the determination of trace amounts of triclosan in river water samples.

Hollow-fiber-supported liquid phase microextraction with in situ derivatization and gas chromatography-mass spectrometry for determination of chlorophenols in human urine samples.

A simple and highly sensitive method that involves hollow-fiber-supported liquid phase microextraction (HF-LPME) with in situ derivatization and gas chromatography-mass spectrometry (GC-MS) was developed for the determination of chlorophenols (CPs) such as 2,4-dichlorophenol (DCP), 2,4,6-trichlorophenol (TrCP), 2,3,4,6-tetrachlorophenol (TeCP) and pentachlorophenol (PCP) in human urine samples. Human urine samples were enzymatically de-conjugated with beta-glucuronidase and sulfatase. After de-conjugation, HF-LPME with in situ derivatization was performed. After extraction, 2 microl of extract was carefully withdrawn into a syringe and injected into the GC-MS system. The limits of detection (S/N=3) and quantification (S/N>10) of CPs in the human urine samples are 0.1-0.2 ng ml(-1) and 0.5-1 ng ml(-1), respectively. The calibration curve for CPs is linear with a correlation coefficient of >0.99 in the range of 0.5-500 ng ml(-1) for DCP and TrCP, and of 1-500 ng ml(-1) for TeCP and PCP, respectively. The average recoveries of CPs (n=6) in human urine samples are 81.0-104.0% (R.S.D.: 1.9-6.6%) with correction using added surrogate standards. When the proposed method was applied to human urine samples, CPs were detected at sub-ng ml(-1) level.

Simultaneous analysis of benzophenone sunscreen compounds in water sample by stir bar sorptive extraction with in situ derivatization and thermal desorption-gas chromatography-mass spectrometry.

A method for the simultaneous measurement of benzophenone (BP) sunscreen compounds, its derivatives 2,4-dihydroxybenzophenone (BP-1), 2-hydroxy-4-methoxybenzophenone (BP-3), 2-hydroxy-4-methoxy-4'-methylbenzophenone (BP-10), 2-hydroxybenzophenone (2OH-BP), 3-hydroxybenzophenone (3OH-BP) and 4-hydroxybenzophenone (4OH-BP), in water samples was developed using stir bar sorptive extraction (SBSE) with in situ derivatization followed by thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS). The detection limit is 0.5-2 ng L(-1) (ppt) for the seven BPs. The method shows good linearity and the correlation coefficients are equal to or higher than 0.990 for all the analyte. The average recoveries of BPs range from 102.0 to 128.1% (RSD<15.4%, n=6). Trace amounts of BPs in river water samples were determined by the present method.

Miniaturized hollow fiber assisted liquid-phase microextraction with in situ derivatization and gas chromatography-mass spectrometry for analysis of bisphenol A in human urine sample.

A new method that involves miniaturized hollow fiber assisted liquid-phase microextraction (HF-LPME) with in situ derivatization and gas chromatography-mass spectrometry (GC-MS) is described for the determination of trace amounts of bisphenol A (BPA) in human urine samples. The detection limit and the quantification limit of BPA in human urine sample are 0.02 and 0.1 ng ml(-1) (ppb), respectively. The calibration curve for BPA is linear with a correlation coefficient of >0.999 in the range of 0.1-50 ng ml(-1). The average recoveries of BPA in human urine samples spiked with 1 and 5 ng ml(-1) BPA are 101.0 (R.S.D.: 6.7%) and 98.8 (R.S.D.: 1.8%), respectively, with correction using the added surrogate standard, bisphenol A-(13)C12. This simple, accurate, sensitive and selective analytical method can be applicable to the determination of trace amounts of BPA in human urine samples.

Determination of benzophenones in river-water samples using drop-based liquid phase microextraction coupled with gas chromatography/mass spectrometry.

In this study, a fast, simple and highly sensitive method that employs liquid phase microextraction (LPME)-GC/MS was developed to analyze trace benzophenones (BPs) in river-water samples. The tip of a 10-microl microsyringe filled with toluene (3 microl) was inserted into 2 ml of a river-water sample, and fixed at 5 mm below the water surface of the sample. A toluene droplet was made on the tip of the syringe, and extraction was conducted while agitating at 500 rpm for 15 min. After extraction, 2.0 microl of the extract was put into the syringe again, and directly introduced to GC/MS. The limits of detection (S/N = 3) and quantification (S/N >10) of BPs were 10 and 50 pg ml(-1), respectively. The results of a recovery test ranged over 93.3 - 101.1% (RSD, less than 10%; n = 6). The results of BPs determinations in the river-water samples showed that BPs (ND - 68.9 pg ml(-1)) were detected.

Stir-bar-sorptive extraction, with in-situ deconjugation, and thermal desorption with in-tube silylation, followed by gas chromatography-mass spectrometry for measurement of urinary 4-nonylphenol and 4-tert-octylphenol glucuronides.

A novel method, stir-bar-sorptive extraction (SBSE), with in-situ deconjugation and thermal desorption (TD) with in-tube silylation, followed by gas chromatography-mass spectrometry (GC-MS), for determination of trace amounts of 4-nonylphenol glucuronide (NP-G) and 4-tert-octylphenol glucuronide (OP-G) in human urine, is described. The method involved correction by use of stable isotopically labeled internal standards 4-(1-methyl)octylphenol-d5 (NP-d) and deuterium 4-tert-octylphenol (OP-d). A human urine sample to which beta-glucuronidase had been added was extracted for 90 min at 37 degrees C using a stir bar coated with a 500-microm-thick layer of polydimethylsiloxane (PDMS). NP-G and OP-G were deconjugated, becoming free 4-nonylphenol (NP) and 4-tert-octylphenol (OP). The analytes were then extracted with the PDMS stir bar and the stir bar was subjected to TD with in-tube silylation; this was followed by GC-MS in selected-ion-monitoring (SIM) mode. To optimize the conditions for SBSE with in-situ deconjugation and to test recovery, NP-G and OP-G were synthesized by a biochemical technique in our laboratory. Average recoveries from human urine samples spiked with NP-G and OP-G were between 91.9 and 95.6% with correction using the added surrogate standards. Limits of detection were 0.11 ng mL-1 for NP and 0.01 ng mL-1 for OP. We also measured background levels of NP-G and OP-G in six urine samples from healthy volunteers. NP and OP were detected in the samples at concentrations of 0.62-1.95 ng mL-1 and <0.04-0.18 ng mL-1, respectively.

Liquid phase microextraction with in situ derivatization for measurement of bisphenol A in river water sample by gas chromatography-mass spectrometry.

A new method that involves liquid phase microextraction (LPME) with in situ derivatization and gas chromatography-mass spectrometry (GC-MS) is described for the determination of trace amounts of bisphenol A (BPA) in river water samples. The LPME conditions, such as the type of extraction solvent and the extraction time, are investigated. Then, the extract is directly injected into GC-MS. The detection limit and the quantification limit of BPA in river water sample are 2 and 10pgml(-1) (ppt), respectively. The calibration curve for BPA is linear with a correlation coefficient of >0.999 in the range of 10-10,000pgml(-1). The average recoveries of BPA in river water samples spiked with 100 and 1000pgml(-1) BPA are 104.1 (RSD: 8.9%) and 98.3 (RSD: 3.2%), respectively, with correction using the added surrogate standard, bisphenol A-(13)C(12). This simple, accurate, sensitive and selective analytical method may be applicable to the determination of trace amounts of BPA in liquid samples.

Dual derivatization-stir bar sorptive extraction--thermal desorption--gas chromatography-mass spectrometry for determination of 17beta-estradiol in water sample.

A novel method for the trace analysis of 17beta-estradiol (E2) in river water sample was developed, which involved stir bar sorptive extraction (SBSE) with in situ acylation (first derivatization) and thermal desorption (TD) with quartz wool assisted (QWA) in tube silylation (second derivatization), followed by gas chromatography-mass spectrometry (GC-MS), and is called the "dual derivatization method." The optimum conditions for SBSE with in situ acylation, such as the volume of acetic acid anhydride and the extraction time, were investigated. In addition, the optimum conditions for TD with QWA in tube silylation, such as the volume of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and the TD temperature and hold time, were investigated as well. The detection limit (S/N = 3) and the quantitation limit (S/N>10) of E2 in the river water sample were 0.5 and 2 pg ml(-1) (ppt), respectively, by the dual derivatization method. In addition, the detection limit was 0.1 pg ml(-1) by using dual derivatization method with multi-shot mode. The calibration curve for E2 was linear in the range of 0.002-10 ng ml(-1) with correlation coefficients >0.999. The average recoveries of E2 (n = 6) at the concentrations of 0.05 and 1.0 ng ml(-1) from the river water sample were 93.1 (RSD: 1.4%) and 98.4% (RSD: 0.8%), respectively, with correction using the added surrogate standard, 17beta-estradiol-(13)C(4). This simple, accurate, sensitive and selective analytical method may be applicable to the determination of trace amounts of E2 in water samples.

Stir bar sorptive extraction with in situ de-conjugation and thermal desorption gas chromatography-mass spectrometry for measurement of 4-nonylphenol glucuronide in human urine sample.

4-Nonylphenol glucuronide (NP-G) in human urine samples was analyzed using stir bar sorptive extraction (SBSE) with in situ de-conjugation by beta-glucuronidase and thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS). Distilled water (1 ml), 1.0 M ammonium acetate solution (100 microl) and beta-glucuronidase (10,000 units ml(-1), 10 microl) were added to human urine sample (1 ml), and extraction was commenced for 90 min at 37 degrees C while stirring at 250 rpm with a stir bar coated with a 500-microm-thick polydimethylsiloxane (PDMS) layer. Then, the stir bar was subjected to TD-GC-MS in the selected ion monitoring (SIM) mode. The calibration curve was made by SBSE method using 4-nonylphenol (NP) as the standard solution. The method showed good linearity and the correlation coefficients were 0.999 over the concentration range of 5-500 nM. Moreover, to optimize the conditions for SBSE with in situ de-conjugation and the recovery test, NP-G was synthesized by a biochemical technique in our laboratory. The limits of detection (S/N = 3) and quantitation (S/N > 10) for NP were 0.2 ng ml(-1) (1.0 nM) and 1.1 ng ml(-1) (5.0 nM), respectively. The average recoveries in the human urine samples (n = 6) spiked with NP-G at levels of 20 and 100 nM were 104.1 (R.S.D. 7.1%) and 100.6% (R.S.D. 9.2%), respectively, with correction using the added internal standard, 4-(1-methyl) octylphenol-d(5). The method enabled the precise determination of the standard and was applicable to the detection of trace amounts of NP-G in human urine samples.