Development of an hollow fiber solid phase microextraction method for the analysis of unbound fraction of imatinib and N-desmethyl imatinib in human plasma.

Therapeutic drug monitoring (TDM) of imatinib (IM) in cancer therapy offers the potential to improve treatment efficacy while minimizing toxicity. There was a significant correlation between unbound concentration and clinical response and toxicity, compared with total plasma concentrations, and the quantification of unbound IM and its metabolite, N-desmethyl imatinib (NDI) are of interest for TDM. However, traditional unbound drug separation methods have shortcomings, especially are susceptible to non-specific binding (NSB) of drugs to the polymer-constructed components of filter membranes, which are difficult to avoid at present. Hence it is necessary to developed a reliable separation method for the analysis of the unbound fraction of IM and NDI in TDM. We developed and validated an hollow fiber solid phase microextraction (HF-SPME) method coupled with high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) that to measure unbound IM and NDI concentration in human plasma. It used the NSB phenomenon and solve the NSB problem. The preparation procedure only involves a common vortex and ultrasonication without dilution of samples and modification of membrane. A total of 50 chronic myeloid leukemia (CML) patients were enrolled in our study. The relationship between the unbound and total concentrations for IM and NDI, as well as the concentration ratios of NDI to IM in 50 clinical plasma samples were investigated. The extraction recovery is high to 95.5-106 % with validation parameters for the methodological results were all excellent. There were both a poor linear relationship between the unbound and total concentrations for IM (r2=0.504) and NDI (r2=0.201) in 50 clinical plasma samples. The unbound concentration ratios of NDI to IM varied widely in CML patients. The determination of unbound IM and NDI concentration is meaningful and necessary. The developed HF-SPME method is simple, accurate and precise that could be used to measure unbound IM and NDI concentration in clinical TDM.

Impact of homocysteine on acute ischemic stroke severity: possible role of aminothiols redox status.

BACKGROUND: Acute ischemic stroke (AIS) is one of the most common cerebrovascular diseases which accompanied by a disruption of aminothiols homeostasis. To explore the relationship of aminothiols with neurologic impairment severity, we investigated four aminothiols, homocysteine (Hcy), cysteine (Cys), cysteinylglycine (CG) and glutathione (GSH) in plasma and its influence on ischemic stroke severity in AIS patients. METHODS: A total of 150 clinical samples from AIS patients were selected for our study. The concentrations of free reduced Hcy (Hcy), own oxidized Hcy (HHcy), free reduced Cys (Cys), own oxidized Cys (cysteine, Cyss), free reduced CG (CG) and free reduced GSH (GSH) were measured by our previously developed hollow fiber centrifugal ultrafiltration (HFCF-UF) method coupled with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The concentration ratio of Hcy to HHcy (Hcy/HHcy), Cys to Cyss (Cys/Cyss) were also calculated. The neurologic impairment severity of AIS was evaluated using National Institutes of Health Stroke Scale (NIHSS). The Spearman correlation coefficient and logistic regression analysis was used to estimate and perform the correlation between Hcy, HHcy, Cys, Cyss, CG, GSH, Hcy/HHcy, Cys/Cyss and total Hcy with NIHSS score. RESULTS: The reduced Hcy and Hcy/HHcy was both negatively correlated with NIHSS score in AIS patients with P = 0.008, r=-0.215 and P = 0.002, r=-0.249, respectively. There was no significant correlation of Cys, CG, GSH, HHcy, Cyss, Cys/Cyss and total Hcy with NIHSS score in AIS patients with P value > 0.05. CONCLUSIONS: The reduced Hcy and Hcy/HHcy, not total Hcy concentration should be used to evaluate neurologic impairment severity of AIS patient.

Distribution and analysis of subacromial spurs and the relationship with acromial classification and angle in healthy individuals.

BACKGROUND: Subacromial spurs are considered the one of the pathology underlying shoulder impingement syndrome. Furthermore, few studies have focused on the morphology of the subacromial spurs in normal Chinese people. This study aimed to study the spur distribution and to illustrate the morphology of spurs, which may help guide the extent of acromioplasty. METHODS: A total of 93 normal individuals were enrolled, and both shoulders of all enrolled individuals were analyzed. The subjects were divided and classified into three different groups by ages: group I = 18-40 years, group II = 41-60 years, and group III ≥ 61 years. The osteophyte distribution, osteophyte area, subacromial surface area and osteophyte area/subacromial surface area ratio were measured and illustrated using Mimics and 3-matic software. The shape of the acromion was classified according to the Bigliani and Morrison classification system. The acromial angle was also classified. Then, the relationship between osteophytes, acromial classification and acromial angle was analyzed. RESULTS: Type II (curved shape) was the most common type of acromion, and the hooked shape was a rare form. A significant increase in the left subacromial surface area in males was observed in group III compared with group I (P < 0.001) and group II (P = 0.004). The total spur/subacromial area ratio was significantly higher in group II than I. An obvious increase in the right subacromial area was observed in group III compared with group I (P = 0.004). Furthermore, there was a significant increase in the right spur area (P = 0.021) and total spur/subacromial area ratio (P = 0.006) in females in group II compared with group I. Fewer spurs were observed on the left than on the right side (p = 0.0482). One spur was most common among type II acromions (29/36) (80.56%) on the left side and the right side (34/52, 65.38%). CONCLUSIONS: Spurs osteophytes are mainly distributed with an irregular shape and mostly run through the medial and lateral sides of the subacromial surface in normal subjects. The characteristics of subacromial spurs are so diverse that a surgeon must conduct subacromial decompression completely based on the morphology of individual spurs.

A more accurate indicator to evaluate oxidative stress in rat plasma with osteoporosis.

Background: oxidative stress is linked to various human diseases which developed into the idea of "disrupted redox signaling". Osteoporosis (OP) is a chronic skeletal disorder characterized by low bone mineral density and deterioration of bone microarchitecture among which estrogen deficiency is the main cause. Lack of estrogen leads to the imbalance between oxidation and anti-oxidation in patients, and oxidative stress is an important link in the pathogenesis of OP. The ratio of the reduced to the oxidized thiols can characterize the redox status. However, few methods have been reported for the simultaneous determination of reduced forms and their oxidized forms of thiols in plasma. Methods: we developed a hollow fiber centrifugal ultrafiltration (HFCF-UF) method for sample preparation and validated a high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method to determine two reduced forms of thiols-homocysteine (Hcy), cysteine (Cys) levels and their respective oxidized compounds, homocystine (HHcy) and cystine (Cyss) in rat plasma simultaneously for the first time. Thirty-six female rats were randomly divided into three groups: normal control (NC), oxidative stress (ovariectomy, OVX) and ovariectomy with hydrogen-rich saline administration (OVX + HRS). Results: the validation parameters for the methodological results were within the acceptance criteria. There were both significant differences of Hcy/HHcy (Hcy reduced/oxidized) and Cys/Cyss (Cys reduced/oxidized) in rat plasma between three groups with both p < 0.05 and meanwhile, the p values of malondialdehyde, superoxide dismutase and glutathione peroxidase were all less than 0.01. The value of both Hcy/HHcy and Cys/Cyss were significantly decreased with the change of Micro-CT scan result of femoral neck in OVX group (both the trabecular thickness and trabecular number significantly decreased with a significant increase of trabecular separation) which demonstrate OP occurs. The change of Hcy/HHcy is more obvious and prominent than Cys/Cyss. Conclusions: the Hcy/HHcy and Cys/Cyss could be suitable biomarkers for oxidative stress and especially Hcy/HHcy is more sensitive. The developed method is simple and accurate. It can be easily applied in clinical research to further evaluate the oxidative stress indicator for disease risk factors.

Accurately quantified plasma free glycine concentration as a biomarker in patients with acute ischemic stroke.

We developed a hollow fiber centrifugal ultrafiltration (HFCF-UF) method to study the change of plasma levels of free glycine (Gly) in patients with acute ischemic stroke (AIS). Twenty-four patients with AIS confirmed by diffusion-weighted imaging (DWI) were enrolled. During the study period, the patients did not receive any supplemental amino acids therapy that could affect the obtained results. Our results showed that although AIS patients adopted different methods of treatment (thrombolytic and non-thrombolytic), the clinical NIHSS score of AIS showed a downward trend whereas Gly concentration showed increased trend. Moreover, plasma free Gly concentration was positively correlated with ASPECTS score. The correlation between Gly levels and infarct volume showed a statistical significance. That is to say, higher Gly level predicted smaller infarct size. Thus, the change of free Gly level in plasma could be considered as a potential biomarker of AIS.

An Analysis of the Morphology of Bony Bicipital Groove Fractures in Proximal Humeral Fractures.

OBJECTIVE: Research on proximal fractures in the humeral bicipital groove (BG), a region in which bones are not commonly fractured, is considered sparse in the literature. The objective of this research was to present the definite characteristics and distribution of BG fractures. METHODS: This retrospective study included and enrolled 119 proximal humeral fractures in adult patients with complete radiography data to identify the fracture distribution in the BG from January 2021 to August 2021. The bicipital region was divided into three parts, i.e. the upper 1/3, middle 1/3, and lower 1/3 of the BG, and the distribution of fracture lines was transcribed on the male or female template, as appropriate. In addition, the normal contralateral humerus was used to calculate the cortical thickness of the supratubercular groove and different parts of the BG (upper, middle, and lower parts). The Mann-Whitney test or one-way ANOVA along with LSD tests were used to determine differences in the fracture distribution and cortical thickness between men and women. RESULTS: Fractures of the BG in both men and women were mainly located in the upper 2/3 region of the BG, especially in the middle 1/3 of the BG. There were significant differences in the cortical thickness of the BG in men compared with that in women. The cortical thickness was highest in the supratubercular ridge but not the BG in men and women, respectively. CONCLUSION: This research concluded that bony BG fractures were always observed in the middle part of the BG and were mainly found in patients with four fractures of the proximal humerus. As a unique fracture pattern, the existence of a bony BG fracture always means that a patient has been injured by a relatively severe mechanism, and more attention should be given to these proximal humeral fractures.

Hydrogen saline water accelerates fracture healing by suppressing autophagy in ovariectomized rats.

Objective: The treatment of osteoporotic fractures is difficult, and to minimize the negative result or poor functional rehabilitation, this study focuses on hydrogen water (HRW) to test its effect on the process of menopausal osteoporotic fracture healing and its relationship with autophagy and to try to reveal the potential mechanism of action of HRW on osteoporotic fractures. Materials and methods: A rat osteoporotic fracture model was established, and HRW was systematically applied with or without 3MA. The results were analyzed with X-rays, micro-CT scans, serum biomarker analysis, biomechanical tests, histopathology, immunohistochemistry, and Western blotting. The sham, OVX, OH (OVX+HRW) and OHA (OVX+HRW+3MA) groups were formed and compared. Results: Increased oxidative stress and autophagy levels were necessary physiological responses in the process of fracture healing. It was found that systemic HRW treatment slightly suppressed autophagy and then activated the Keap1-Nrf2 signaling pathway by maintaining the Keap1-Nrf2-P62 interaction and improved the osteoporotic fracture healing process. Conclusion: HRW treatment activated the Keap1-Nrf2 signaling pathway to antagonize cellular stress by suppressing autophagy levels, especially at the early stage of the fracture healing process, and this was beneficial to osteoporotic fracture healing in rats.

Tight junction disruption through activation of the PI3K/AKT pathways in the skin contributes to blister fluid formation after severe tibial plateau fracture.

Background: Acute compartment syndrome (ACS) is an orthopedic emergency that commonly occurs after severe tibial plateau fracture. Fracture blisters form on the skin, and it was found in our previous study that when blisters form, the compartment pressure significantly decreases. However, the potential mechanism underlying this pressure decrease has not yet been elucidated. Methods: To obtain a comprehensive understanding of the changes that occur after blister formation on the skin, the changes in tight junction expression in the skin after tibial plateau fracture were observed. Blister samples and normal skin were collected from patients with bicondylar tibial plateau fractures with or without blisters. The epidermis thickness was measured, and the difference in the levels of K1, K5, K10, and skin barrier proteins such as claudin 1, claudin 2, and occludin between the two groups was evaluated by immunochemistry analysis, immunofluorescence, Western blotting, and qPCR. Results: The skin was thinner and the levels of K1, K5, and K10 were significantly decreased in blistered skin. Furthermore, the PI3K/AKT pathway was found to be activated, and the tight junction expression was significantly decreased in blistered skin. This indicates that the paracellular pathway, which is essential for accelerating fluid accumulation in blisters and indirectly decreases compartment pressure, was activated. Conclusion: Changes in the tight junction expression after blister formation may underlie blister fluid formation and indirectly explain the decrease in compartment pressure under blistered skin after severe tibial plateau fracture.

The Reliability and Reproducibility of a New Revised Edelson Classification.

OBJECTIVE: Edelson classification is a 3D classification of proximal humeral fractures, but there is a scarcity of application of this classification in large samples, and the accuracy of classification was also not testified. The objective of this research was to verify whether a revised Edelson classification produces satisfactory agreement for proximal humeral fracture classification in adult patients. METHODS: A total of 827 proximal humeral fractures (304 male and 520 female patients, 58.0 ± 16.2 years) were found retrospectively from January 2014 to December 2019, and classified according to the traditional and newly proposed Edelson classification. The three-dimensional CT images were processed, rotated and visualized within software. Five shoulder surgeons classified each fracture. After data collection, radiographic classifications results were compared by inter- and intraobserver analysis with the method of weighted kappa coefficients. Fracture classification based on Edelson and revised Edelson classification was presented and compared. RESULTS: The mean k value for the interobserver reliability was 0.748 (range, 0.583 to 0.958) compared with Edelson classification (0.548, range, 0.48 to 0.635), indicating satisfactory agreement. The mean k value for intraobserver reliability was 0.906 (range, 0.823 to 0.943) compared with Edelson classification (0.762, range, 0.666 to 0.808), indicating excellent agreement when using the newly revised Edelson classification. The mechanism was categorized as the shoulder being in a position of forward flexion, abduction, and internal rotation in Edelson I-IV and bicipital fractures. For the greater tuberosity fracture, the mechanism was classified into two mechanisms based on the presence of a combined dislocation. Bicipital groove fracture is a commonly observed fracture pattern, and included in the revised Edelson classification. CONCLUSIONS: The revised Edelson classification proposed was more in line with the injury mechanism of the fracture, was beneficial in identifying more fracture types such as bicipital groove fracture, and verified to be a good proximal humeral fracture classification with good reliability compared with the traditional Edelson classification.

Analysis of free concentrations of lamotrigine and active oxcarbazepine metabolite in clinical patients by hollow-fiber centrifugal ultrafiltration.

Aim: To establish a simple and accurate method to explore the correlation between free and total concentrations of lamotrigine (LTG) and the active oxcarbazepine metabolite monohydroxy derivative (MHD) (10,11-dihydro-10-hydroxycarbamazepine) in clinical patients. Materials & methods: Serum samples were prepared by hollow-fiber centrifugal ultrafiltration and then injected into UPLC for analysis. Results: Absolute recovery was as high as approximately 90.1-98.6% with excellent precision (relative standard deviation <6.7%). Analysis time was reduced to 5 min. There were significant individual differences in the protein binding rates of both LTG and MHD that were probably due to the use of different clinical patients. Conclusion: Free concentrations of LTG and MHD cannot be estimated by total concentration in specific clinical patients. Free drug monitoring of LTG and MHD in clinical therapeutic drug monitoring is important and essential.

Norvancomycin for the treatment of central nervous system MRSA infections: A randomized controlled trial.

Combined intravenous and intrathecal administration of norvancomycin (NVCM) is routinely employed in treating methicillin-resistant Staphylococcus aureus (MRSA) ventriculitis in patients following craniotomy. However, the optimal dosing regimen, the pharmacokinetics (PK) of NVCM in cerebrospinal fluid (CSF), and the clinical outcome are yet to be elucidated. Herein, a single-center randomized controlled trial was conducted in the Neurosurgery Department of the Second Hospital of Hebei Medical University (Shijiazhuang, China). Patients with MRSA ventriculitis after craniotomy were randomly assigned to two groups. The control group received 800 mg NVCM intravenously every 12 h, and the experimental group received 800 mg NVCM intravenously every 12 h and 16 mg NVCM intrathecal administration every 24 h. The primary outcome was the length of therapy, while the secondary outcomes included the area under the concentration-time curve in 0-24 h/minimum inhibitory concentration ratio (AUC0-24h/MIC) of NVCM in CSF. A total of 29 patients (14 in the experimental group and 15 in the control group) were included in this study. Of these, 24 constituted the final analysis population, with 12 in each group. The average length of therapy in the experimental group was markedly shorter than that of the control group (11.2 ± 2.6 days vs. 16.6 ± 5.2 days, P = 0.005), while the AUC0-24h/MIC in the experimental group was significantly higher than that in the control group (2306.57 ± 928.58 vs. 46.83 ± 27.48, P < 0.001) with no increase in adverse reactions. Combined intravenous and intrathecal administration can shorten the treatment time of intracranial infection without higher adverse reaction risks in our research. Further studies with larger sample size are warranted to verify its safety and efficacy.

Morphological characteristics of the surgical neck region in the proximal humerus at different ages.

BACKGROUND: The objective of the study was to demonstrate the cortical thickness character in the humeral surgical neck region using 3D cortical bone mapping technique and try to illustrate its morphological changes with age. MATERIAL AND METHODS: Normal individuals, including 11 volunteers younger than 18 years, 87 adult men and 46 adult women, were enrolled. The cortical thickness and height of the surgical neck region was measured with Mimic and 3 Matic software. The height of the region was compared and measured. People with an age of 18-30 years was identified as Group I, people in 31-40 years as Group II, people in 41-50 years as Group III, people in 51-60 years as Group IV, and Group V including people ≥ 61 years. RESULTS: Compared with the baseline Group I, cortical thickness was significantly decreased by 0.52 mm (P = 0.006) in Group III, by 0.76 mm (P < 0.001) in Group IV, and by 0.77 mm (P < 0.001) in Group V. Age moderately predicted cortical thickness with r = -0.5481. The height of the cortical change region was significantly decreased by 2.25 mm (P = 0.007) in Group II, by 2.98 mm (P < 0.001) in Group III, and by 2.07 mm (P = 0.02) in Group IV. However, no significant decrease was illustrated in Group V (0.57 mm) (P = 0.891). The relation between age and the height of the cortical thickness change region was nonlinear. CONCLUSIONS: This study identified an obvious decrease in cortical thickness with aging, and the height was curve fitted with aging in surgical neck region.

An exploratory study of pelvis anatomy to revise the bony canal used for LC2 screw insertion.

BACKGROUND: Percutaneous screw placement, especially the insertion of LC2 screws, is technically demanding. Although the traditional LC2 bony canal spans the anterior inferior iliac spine (AIIS) to the posterior superior iliac spines (PSIS), a high perforation rate has been reported. OBJECTION: The aim of this research was to design a revised bony canal, measure the canal width and length and guide the insertion of LC2 screws for pelvic fractures. MATERIALS AND METHODS: The plane tool in the Mimics analysis menu was used to draw a midplane connecting the midpoint between the anterior inferior spine and the PSIS upper flat region with pelvic CT data. The minimum widths of the upper, middle, lower surfaces of the tunnel and perforation rate were measured and compared. The ideal screw length was also measured along the longitudinal axis running through the midpoint of the midplane. RESULTS: The minimum widths of the upper, middle and lower surfaces of the revised canal were 3.63 mm, 7.7 mm, and 11.93 mm, respectively, in males and 5.97 mm, 9.93 mm, and 12.45 mm, respectively, in females. Significant differences were observed among the upper, middle and lower surfaces of the revised canal in male patients (P < 0.001). In female subjects, the upper canal surface was significantly different from the middle and lower canal surfaces (P < 0.001). The perforation rate was significantly decreased especially in females pelvic. The channel length passing through the midpoint of the narrowest position of the pelvis was 130.85 ± 8.02 mm in males and 124.30 ± 7.71 mm in females and was significantly different for male and female pelvises (P = 0.004). CONCLUSION: The LC2 screw should be inserted along the intersection line of the AIIS lateral wall and the iliac body. The screw should be inserted under the line between the midpoint of the AIIS and the PSIS upper flat region to ensure accuracy of placement. LC2 screws can be more easily inserted in males than in females, and the rate of cortical perforation can be significantly decreased under the guidance of the newly proposed canal.

Differences in fixation to young and elderly quadrilateral surfaces with anatomic quadrilateral surface plate (AQSP) based on cortical thickness morphological results.

AIMS: With relatively thinner cortical thickness, the management of acetabulum osteoporotic fractures in elderly patients is difficult. The aim of the research was to compare and present the morphological characteristics of the quadrilateral plate in young and elderly age groups, such as the area, and position distribution of the thin cortical thickness region, fracture lines maps, and propose a revised design plate for elderly patients based on these anatomic information. METHODS: As a retrospective research, acetabular fracture with one normal hemipelvises, including 110 men and 39 women, were collected to present the morphological characteristics of the quadrilateral region. The subjects were divided into three different age groups: Group I = 18-40 years (31.3 ± 6.6 years), Group II = 41-60 years (49.9 ± 5.3 years), and Group III ≥ 61 years (68.7 ± 6.8 years). The area of the quadrilateral surface, the area and position distribution of the thin cortical thickness region, the ratio and fracture lines maps were calculated and compared with Mimics in different groups. RESULTS: The thin cortical thickness/width region area (TCWRA) was significantly increased in Group III compared with Group I and Group II. The ratio of TCWRA accounted for in the quadrilateral region was also significantly increased in Group III (≥ 61 years) compared with Group I (P = 0.01) and Group II (P = 0.011). None of the subjects had a component involving the "A" zone, thirty-three thin cortical thickness regions were located in the "B" zone, and one hundred and sixteen involved both zones of the quadrilateral plate ("A + B" zone). Furthermore, there were a significant differences in the fracture line distributions in three age groups. More fracture lines of elderly patients were located at anterior part (B zone) compared with Group I and Group II. CONCLUSIONS: It was identified the area of thin cortical thickness region increased as age grown, and fracture lines were inclined to be more distributed in "B" zone in elderly patients. To meet the demands of acetabular fixation in different age groups, cortical thickness changes in young and elderly individuals should be given special attention when the quadrilateral surface plate is designed. Level of evidence Level IV, observational study.

Development of silica molecularly imprinted polymer on carbon dots as a fluorescence probe for selective and sensitive determination of cetirizine in saliva and urine.

A fluorescence probe based on carbon dots (CDs) coated with silica molecularly imprinted polymer (MIPs) was synthesized for selective and sensitive determination of cetirizine (CTZ). Green source carbon dots were firstly derived from orange peels through a microwave method, and had the merits of eco-friendly and low toxicity. Then a thin silica film was formed on the surface of CDs by reverse microemulsion technique, and molecularly imprinted polymer coated on silica-carbon dots. In this scene, CTZ, 3-aminopropyltriethoxysilane (APTES) and tetraethoxysilane (TEOS) were employed as a template, a functional monomer and cross linker, respectively. The obtained CDs-MIPs can selectively bind CTZ through the specific interaction between recognition sites and template, and obey photoinduced electron transfer fluorescence quenching mechanism. Fluorescence dropped linearly in the range of 0.5-500 ng mL-1, under the optimal conditions, with a detection limit of 0.41 ng mL-1. Furthermore, the proposed method was successfully intended for the determination of trace CTZ in human saliva and urine samples without the interference of other molecules and ions. And recoveries ranged from 95.8% to 99.8% with relative standard deviation less than 3.0%.

A simple and accurate HFCF-UF method for the analysis of homocysteine, cysteine, cysteinyl-glycine, and glutathione in human blood.

The presence of reduced aminothiols, including homocysteine (Hcy), cysteine (Cys), cysteinyl-glycine (CG), and glutathione (GSH), is significantly increased in the pathological state. However, there have been no reports on the relationship between reduced aminothiols (Hcy, Cys, CG, and GSH) and different genders, ages, and drug combinations in human blood. The accurate quantification of these reduced thiols in biological fluids is important for monitoring some special pathological conditions of humans. However, the published methods typically not only require cumbersome and technically challenging processing procedures to ensure reliable measurements, but are also laborious and time-consuming, which may disturb the initial physiological balance and lead to inaccurate results. We developed a hollow fiber centrifugal ultrafiltration (HFCF-UF) method for sample preparation coupled with a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method and used it to determine four reduced aminothiols (Hcy, Cys, CG, and GSH) in human blood for the first time. A total of 96 clinical patients were enrolled in our study. The influence of different genders, ages, and drug combinations on the levels of four reduced thiols in human blood was also discussed by SPSS 24.0. The sample preparation was simplified to a single 5 min centrifugation step in a sealed system that did not disturb the physiological environment. The validation parameters for the methodological results were excellent. The procedure was successfully applied to monitoring the concentrations of four reduced aminothiols (Hcy, Cys, CG, and GSH) in 96 clinical blood samples. There were no significant differences in Hcy, Cys, CG, or GSH for the different genders, ages, or combinations with methotrexate or vancomycin (P > 0.05). However, there was a significant increase in Hcy concentration in patients treated with valproic acid who were diagnosed with epilepsy (p=0.0007). It is advisable to measure reduced Hcy level in patients taking valproic acid. The developed HFCF-UF method was simple and accurate. It can be easily applied in clinical research to evaluate oxidative stress in further study.

Relationship Between the Free and Total Methotrexate Plasma Concentration in Children and Application to Predict the Toxicity of HD-MTX.

High-dose methotrexate (HD-MTX) can be highly effective as well as extremely toxic. Many drug molecules can bind to plasma proteins to different extents in vivo, whereas only the free drug can reach the site of action to exert a pharmacological effect and cause toxicity. However, free MTX concentrations in plasma have not been reported. Traditional analyses of free drugs are both cumbersome and inaccurate. We collected 92 plasma samples from 52 children diagnosed with ALL or NHL or other lymphomas that were treated with HD-MTX. The hollow fiber centrifugal ultrafiltration (HFCF-UF) was used to prepare plasma samples for analysis of the free MTX concentration. Protein precipitation was employed to measure the total MTX concentration. The HFCF-UF is a simple method involving a step of ordinary centrifugation; the validation parameters for the methodological results were satisfactory and fell within the acceptance criteria. A linearity coefficient r 2 of 0.910 was obtained for the correlation between the free and total MTX plasma concentrations in 92 plasma samples. However, the free and total MTX concentrations was only weakly correlated in 16 clinical plasma specimens with total MTX concentrations >2 µmol L-1 (r 2 = 0.760). Both the free and total MTX concentrations at 42 h were negatively correlated with the creatinine clearance (CCr) level (P = 0.023, r = -0.236 for total MTX and P = 0.020, r = -0.241for free MTX, respectively). The free MTX concentration could not be accurately estimated from the total MTX concentration for patients with high MTX levels which are conditions under which toxic reactions are more likely to occur. High plasma MTX levels could become a predictor of the occurrence of MTX nephrotoxicity to draw people's attention. The proposed HFCF-UF method is a simple and accurate way to evaluate efficacy and toxicity in clinical therapeutic drug monitoring.

Determination of Free Valproic Acid Concentration in 569 Clinical Samples by LC-MS/MS After Hollow Fiber Centrifugal Ultrafiltration Treatment.

OBJECTIVE: To perform therapeutic drug monitoring of total and free plasma valproic acid (VPA) concentrations in clinical samples and to analyze the related factors. METHODS: The total VPA concentration in plasma was determined by ultrahigh-performance liquid chromatography with precolumn derivatization with α-bromoacetophenone, and the free VPA concentration was determined by liquid chromatography-tandem mass spectrometry after the plasma was treated by hollow fiber centrifugal ultrafiltration. Regression analysis was performed to examine the associations between free plasma VPA, total plasma VPA, and the plasma protein binding rate. The impact of individual situations, outpatient or inpatient factors, and drug combinations on VPA concentrations were examined. RESULTS: Of the 569 clinical samples, 268 were inpatients and 301 were outpatients, and the total VPA concentration in 138 cases (24.2%) was lower than the effective treatment concentration range; the total and free VPA concentrations in outpatient samples were 11.0% and 26.1% higher than those of inpatients, respectively. There was no linear relationship between the free and total VPA concentrations. The relationship equation between the plasma protein binding rate and free VPA concentrations was as follows: Y = 0.0255X2 - 1.1357X + 97.429 (r = 0.8011). The total and free VPA concentrations were significantly decreased after the coadministration of phenobarbital (83.7% and 64.3% of the control group, P < 0.05) or carbapenem antibiotics (32.0% and 32.7% of the control group, P < 0.05). CONCLUSIONS: The total VPA concentrations in patients with epilepsy at our hospital was lower than the effective treatment concentration range, which was inadequate for epilepsy control; the total VPA concentrations of outpatients were higher than those of inpatients; as phenobarbital affects VPA metabolism, therapeutic drug monitoring is recommended. Carbapenem antibiotic coadministration with VPA should be avoided because carbapenem antibiotics can lead to the failure of VPA antiepileptic treatment.

CYP2C9*3/*3 Gene Expression Affects the Total and Free Concentrations of Valproic Acid in Pediatric Patients with Epilepsy.

PURPOSE: To perform therapeutic drug monitoring (TDM) of total and free plasma valproic acid (VPA) concentrations in pediatric patients with epilepsy and to analyze related factors. PATIENTS AND METHODS: Pediatric epileptic patients treated in 2015-2019 in our hospital were assessed. Total and free plasma VPA concentrations were obtained by UPLC and LC-MS/MS, respectively. Regression analysis was performed to examine the associations of free plasma VPA with total plasma VPA and plasma protein binding rate. The impacts of individual situation, CYP2C9 genotype, and drug combination on VPA concentration were examined. RESULTS: Of the 251 patients, 81 had lower total concentrations than effective therapeutic levels; 86 and 31 patients had infections and central nervous system dysplasia, respectively. VPA's daily doses and free drug concentrations were significantly lower in the CYP2C9 *3/*3 genotype group versus the CYP2C9 *1/*3 and CYP2C9 *1/*1 groups (P<0.05). Free and total VPA concentrations were linked by Y = 0.0004 X2 + 0.042 X + 0.3035 (r=0.6981); VPA plasma protein binding rate and free VPA concentration were related by Y = 0.0003 X2 - 0.0127 X + 0.9777 (r=0.8136). Both total and free VPA concentrations were significantly decreased in patients simultaneously administered phenobarbital, meropenem and biapenem (P<0.05), with therapeutic failure after meropenem/biapenem co-administration. CONCLUSION: Free VPA amounts have nonlinear relationships with total VPA amounts and plasma protein binding rate in epileptic children. Additionally, CYP2C9 *3/*3 expression affects VPA metabolism. Since phenobarbital affects VPA metabolism, TDM is recommended. Meanwhile, carbapenem-co-administration with VPA should be prohibited.

A Direct Injection Technique to Improve Biosafety to Analyze Levetiracetam Concentrations in Human Serum and Its Application in Therapeutic Drug Monitoring.

BACKGROUND: With the outbreak of COVID-19, it has become very important to improve biosafety measures taken by medical staff. Fewer pretreatment steps correspond to lower chances of infection. The authors established a direct injection technique to analyze levetiracetam (LEV) concentrations in human serum and studied its application in therapeutic drug monitoring. METHODS: Serum samples were prepared by hollow fiber centrifugal ultrafiltration and the filtrate was directly injected into a ultra-high performance liquid chromatography apparatus (Waters UPLC BEH C18 column: 50 × 2.1 mm, 1.7 µm) for analysis. The mobile phase consisted of acetonitrile and water (8:92) at a flow rate of 1.0 mL/min. The column temperature was maintained at 30°C. The detected wavelength was 210 nm. RESULTS: A linear relationship was obtained for LEV from 0.625 to 80 mcg/mL (r2 = 0.999). The limit of detection for the analysis of LEV was 0.125 mcg/mL. The analysis time was shortened to 4 minutes. The recovery rate of LEV based on the current method was 96.6%-100.1%, whereas the absolute recovery rate was 93.2%-96.8%. The relative SD of intraday and interday precision was <7.3%. Stability was achieved at room temperature for 24 hours after 3 freeze-thaw cycles and at -80°C for 21 days. The method was successfully applied to determine LEV concentrations in the serum of 19 patients. CONCLUSIONS: The present method is simple, accurate, and sensitive, and can improve biosafety with the direct injection technique. It is suitable for the analysis of LEV concentrations in therapeutic drug monitoring.