Bilateral efforts to improve SERS detection efficiency of exosomes by Au/Na7PMo11O39 Combined with Phospholipid Epitope Imprinting.

Detection of cancer-related exosomes in body fluids has become a revolutionary strategy for early cancer diagnosis and prognosis prediction. We have developed a two-step targeting detection method, termed PS-MIPs-NELISA SERS, for rapid and highly sensitive exosomes detection. In the first step, a phospholipid polar site imprinting strategy was employed using magnetic PS-MIPs (phospholipids-molecularly imprinted polymers) to selectively isolate and enrich all exosomes from urine samples. In the second step, a nanozyme-linked immunosorbent assay (NELISA) technique was utilized. We constructed Au/Na7PMo11O39 nanoparticles (NPs) with both surface-enhanced Raman scattering (SERS) property and peroxidase catalytic activity, followed by the immobilization of CD9 antibodies on the surface of Au/Na7PMo11O39 NPs. The Au/Na7PMo11O39-CD9 antibody complexes were then used to recognize CD9 proteins on the surface of exosomes enriched by magnetic PS-MIPs. Lastly, the high sensitivity detection of exosomes was achieved indirectly via the SERS activity and peroxidase-like activity of Au/Na7PMo11O39 NPs. The quantity of exosomes in urine samples from pancreatic cancer patients obtained by the PS-MIPs-NELISA SERS technique showed a linear relationship with the SERS intensity in the range of 6.21 × 107-2.81 × 108 particles/mL, with a limit of detection (LOD) of 5.82 × 107 particles/mL. The SERS signal intensity of exosomes in urine samples from pancreatic cancer patients was higher than that of healthy volunteers. This bidirectional MIPs-NELISA-SERS approach enables noninvasive, highly sensitive, and rapid detection of cancer, facilitating the monitoring of disease progression during treatment and opening up a new avenue for rapid early cancer screening.

Comparative evaluation of the extraction and analysis of urinary phospholipids and lysophospholipids using MALDI-TOF/MS.

Lipids, particularly phospholipids (PLs) and lysophospholipids (LPLs), are attracting increasing scientific interest for their biological functions in cells and their potential as disease biomarkers for Alzheimer's disease and several types of cancer. Urinary PLs and LPLs could be ideal clinical biomarkers, because urine can be collected easily and noninvasively. However, due to their very low concentrations in urine compared with the relatively large quantity of contaminants in this matrix, efficient extraction and sensitive detection are required for analyzing urinary PLs and LPLs. In this study, various methods for analyzing PLs and LPLs in urine were compared and optimized from a clinical perspective. An optimized lipid extraction method and a matrix for matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) were established using two external ionization standards and an internal standard mix containing 13 human urinary lipids. 9-Aminoacridine (9-AA) was a useful and effective matrix for the MALDI-TOF/MS analysis of all the internal standard lipids in both positive and negative ion modes. However, it was necessary to determine the proportional lipid concentrations from the balance between the extracted lipid and the matrix. The extraction efficiency and reproducibility of the acidified Bligh and Dyer method were excellent for both positively and negatively charged lipids. Analysis of small volumes of urine was the most efficient with the 9-AA MALDI matrix at concentrations of or below 5 mM. The combined analytical procedures allowed rapid and comprehensive screening of low concentrations of PLs and LPLs in clinical samples.

A multiplexed UPLC-MS/MS assay for the simultaneous measurement of urinary safety biomarkers of drug-induced kidney injury and phospholipidosis.

Drug-induced kidney injury (DIKI) is a major concern in drug risk assessment given its clinical importance and the absence of a sensitive/specific method of diagnosis. Pharmaceutical regulatory agencies have qualified and issued letters of support for new biomarkers to better evaluate DIKI in nonclinical toxicity and clinical studies. Additional efforts have focused on drug-induced phospholipidosis (DIPL) and its potential link with collateral renal damage. The combined use of urinary biomarkers is an efficient way to evaluate renal safety in nonclinical and clinical studies. Eight FDA/EMA/PMDA qualified (or supported) urinary biomarkers, including kidney injury molecule-1 (KIM-1), ß2-microglobulin (B2M), clusterin (CLU), cystatin C (CysC), trefoil factor 3 (TFF3), neutrophil gelatinase-associated lipocalin (NGAL), osteopontin (OPN), and alpha-glutathione S-transferase (α-GST), were quantified by multiplex UPLC-MS/MS in a repeat dose study of gentamicin in rats. Rats administered gentamicin at 100 mg/kg/day for 2 weeks developed renal lesions detected by histopathology. Biomarkers of tubular damage (CLU, KIM-1, OPN) increased 9.8, 34.7, and 35.6-fold (relative to concurrent controls), respectively, after 2 weeks of dosing. Biomarkers of glomerular damage and/or impairment of tubular reabsorption (CysC, B2M) increased 11.7 and 22.6-fold. NGAL and α-GST increased <3-fold after 2 weeks of dosing. TFF3 was comparable to concurrent controls. The elevated biomarker concentrations met PSTC threshold criteria and were consistent with mechanisms of gentamicin nephrotoxicity. Increased urinary di-22:6-BMP indicated concomitant DIPL as confirmed by TEM. This work provides evidence supporting the combined use of the DIKI biomarker panel and di-22:6-BMP as a biomarker of DIPL in drug risk assessment.

Liposomes as potential masking agents in sport doping. Part 1: analysis of phospholipids and sphingomyelins in drugs and biological fluids by aqueous normal-phase liquid chromatography-tandem mass spectrometry.

In the present work, aqueous normal-phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), in different acquisition modes, was employed for the direct analysis and profiling of nine phospholipid classes (phosphatidic acids, phosphatidylserines, phosphatidylethanolamines, lysophosphatidylethanolamines, phosphatidylglycerols, phosphatidylinositols, phosphatidylcholines, lysophosphatidylcholines, and sphingomyelins) in biological and pharmaceutical matrices. After chromatographic separation by a diol column, detection and elucidation of phospholipid and sphingomyelin classes and molecular species were performed by different scan acquisition modes. For screening analysis, molecular ions [M + H]+ were detected in positive precursor ion scan of m/z 184 for the classes of phosphatidylcholines, lyso-phosphatidylcholines and sphingomyelins; while phosphatidylethanolamines and lyso-phosphatidylethanolamines were detected monitoring neutral loss scan of 141 Da; and phosphatidylserines detected using neutral loss scan of 184 Da. Molecular ions [M-H]- were instead acquired in negative precursor ion scan of m/z 153 for the classes of phosphatidic acids and phosphatidylglycerols; and of m/z 241 for the phosphatidylinositols. For the identification of the single molecular species, product ion scan mass spectra of the [M + HCOO]- ions for phosphatidylcholines and [M + H]+ ions for the other phospholipids considered were determined for each class and compared with the fragmentation pattern of model phospholipid reference standard. By this approach, nearly 100 phospholipids and sphingomyelins were detected and identified. The optimized method was then used to characterize the phospholipid and sphingomyelin profiles in human plasma and urine samples and in two phospholipid-based pharmaceutical formulations, proving that it also allows to discriminate compounds of endogenous origin from those resulting from the intake of pharmaceutical products containing phospholipidic liposomes. Copyright © 2016 John Wiley & Sons, Ltd.

Cross-Classification of Human Urinary Lipidome by Sex, Age, and Body Mass Index.

Technological advancements in past decades have led to the development of integrative analytical approaches to lipidomics, such as liquid chromatography-mass spectrometry (LC/MS), and information about biogenic lipids is rapidly accumulating. Although several cohort-based studies have been conducted on the composition of urinary lipidome, the data on urinary lipids cross-classified by sex, age, and body mass index (BMI) are insufficient to screen for various abnormalities. To promote the development of urinary lipid metabolome-based diagnostic assay, we analyzed 60 urine samples from healthy white adults (young (c.a., 30 years) and old (c.a., 60 years) men/women) using LC/MS. Women had a higher urinary concentration of omega-3 12-lipoxygenase (LOX)-generated oxylipins with anti-inflammatory activity compared to men. In addition, young women showed increased abundance of poly-unsaturated fatty acids (PUFAs) and cytochrome P450 (P450)-produced oxylipins with anti-hypertensive activity compared with young men, whereas elderly women exhibited higher concentration of 5-LOX-generated anti-inflammatory oxylipins than elderly men. There were no significant differences in urinary oxylipin levels between young and old subjects or between subjects with low and high BMI. Our findings suggest that sex, but neither ages nor BMI could be a confounding factor for measuring the composition of urinary lipid metabolites in the healthy population. The information showed contribute to the development of reliable biomarker findings from urine.

Juvenile Male Rats Exposed to a Low-Dose Mixture of Twenty-Seven Environmental Chemicals Display Adverse Health Effects.

Humans are exposed to a large number of environmental chemicals in their daily life, many of which are readily detectable in blood or urine. It remains uncertain if these chemicals can cause adverse health effects when present together at low doses. In this study we have tested whether a mixture of 27 chemicals administered orally to juvenile male rats for three months could leave a pathophysiological footprint. The mixture contained metals, perfluorinated compounds, PCB, dioxins, pesticides, heterocyclic amines, phthalate, PAHs and others, with a combined dose of 0.16 (Low dose), 0.47 (Mid dose) or 1.6 (High dose) mg/kg bw/day. The lowest dose was designed with the aim of obtaining plasma or urine concentrations in rats at levels approaching those observed in humans. Some single congeners were administered at doses representative of combined doses for chemical groups. With this baseline, we found effects on weight, histology and gene expression in the liver, as well as changes to the blood plasma metabolome in all exposure groups, including low-dose. Additional adverse effects were observed in the higher dosed groups, including enlarged kidneys and alterations to the metabolome. No significant effects on reproductive parameters were observed.

On-line high speed lipid extraction for nanoflow liquid chromatography-tandem mass spectrometry.

An on-line lipid extraction method is introduced by utilizing a short capillary extraction column using HILIC and C4 particles prior to nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS/MS). The on-line extraction using a urine sample spiked with PL standards showed similar or slightly higher recovery values (86%-96%) of phospholipids (PLs) compared to those obtained by the conventional off-line extraction based on the Folch method with or without using the air-exposed drying process. In this study, we demonstrated that PL oxidation can occur during the air-exposed drying process of lipid extracts in standard liquid-liquid extraction procedures, which was confirmed by the oxidized PL (OxPL) molecules that were generated from an off-line extraction using a few PL standards. Quantitative comparison of these OxPL species between on- and off-line extraction followed by nLC-MS/MS with multiple reaction monitoring (MRM) analysis showed a significant decrease (2-10 fold) in unwanted OxPL species when on-line extraction was employed. While the number of identified PLs from a urine sample was somewhat lower than those by off-line extraction, the number of OxPLs was significantly reduced (from 70 to 22) with on-line extraction. The new method offers high speed (∼5min) automated extraction of PLs with nLC-MS/MS analysis and presents the possibility of handling a biological sample with a very limited amount of lipids.

Changes of urinary phospholipids in the chronic kidney disease patients.

OBJECTIVE: To evaluate whether urinary phospholipids could be regarded as biomarkers of chronic kidney disease. MATERIALS AND METHODS: Thirteen healthy volunteers and 26 consecutive chronic kidney disease patients were included. Urinary phospholipids were quantified by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. RESULTS: Urinary phosphatidylcholines concentrations (PC 16:0/16:0, 16:0/22:3, 16:0/18:1 and 16:0/18:2) were significantly higher both in glomerulonephritis group (all p < 0.001) and in tubulointerstitial injury group (all p < 0.05) than in healthy control group. Meanwhile, sphingomyelin concentrations (SM 18:1/16:0 and 18:1/18:0) in glomerulonephritis group were significantly higher than those in healthy control group (all p < 0.001). Urinary PCs and SMs were positively correlated with proteinuria but negatively correlated with serum albumin. Meanwhile, PCs were positively correlated with serum creatinine. CONCLUSION: Our work first demonstrated that urinary phospholipids might be biomarkers for the chronic kidney disease patients. Increased urinary phospholipids in chronic kidney disease patients might result from proteinuria, damaged kidney function or proteinuria induced hypoalbuminemia or lipotoxicity.

Computational approach to structural identification of phospholipids using raw mass spectra from nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry.

A qualitative analysis tool (LiPilot) for identifying phospholipids (PLs), including lysophospholipids (LPLs), from biological mixtures is introduced. The developed algorithm utilizes raw data obtained from nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry experiments of lipid mixture samples including retention time and m/z values of precursor and fragment ions from data-dependent, collision-induced dissociation. Library files based on typical fragmentation patterns of PLs generated with an LTQ-Velos ion trap mass spectrometer are used to identify PL or LPL species by comparing experimental fragment ions with typical fragment ions in the library file. Identification is aided by calculating a confidence score developed in our laboratory to maximize identification efficiency. Analysis includes the influence of total ion intensities of matched and unmatched fragment ions, the difference in m/z values between observed and theoretical fragment ions, and a weighting factor used to differentiate regioisomers through data filtration. The present study focused on targeted identification of particular PL classes. The identification software was evaluated using a mixture of 24 PL and LPL standards. The software was further tested with a human urinary PL mixture sample, with 93 PLs and 22 LPLs identified.

Comparison of urinary and serum levels of di-22:6-bis(monoacylglycerol)phosphate as noninvasive biomarkers of phospholipidosis in rats.

Phospholipidosis (PLD), an abnormal accumulation of phospholipids within tissues, is observed during the preclinical testing of many pharmaceutical drugs. Diagnosis of PLD is currently based on morphologic criteria. An understanding of the clinical incidence of PLD and its possible relationship to adverse drug reactions has been hampered by the absence of noninvasive biomarkers for PLD. The uncommon phospholipid di-docosahexaenoyl bis(monoacylglycerol) phosphate (di-22:6-BMP) has been proposed as a potential urinary biomarker for PLD, but data on the utility of serum di-22:6-BMP measurements in diagnosing PLD is more limited. In this report, we compared the performance of serum and urinary di-22:6-BMP as biomarkers for PLD in rats treated with the PLD-inducing drugs amiodarone and 4,4'-diethylaminoethoxyhexestrol dihydrochloride or the hepatotoxicant acetaminophen (APAP). Serum levels of di-22:6-BMP showed a higher correlation to a generalized PLD incidence score than did levels in urine, but were unexpectedly elevated in rats with marked levels of APAP-induced liver necrosis. When samples were filtered based on serum ALT or liver histopathology thresholds, the diagnostic accuracy of serum di-22:6-BMP for PLD improved to the high level observed for urinary di-22:6-BMP without sample exclusion. These data help define the potential context-of-use of serum di-22:6-BMP as a non-clinical biomarker of PLD.

Effect of ionization modifiers on the simultaneous analysis of all classes of phospholipids by nanoflow liquid chromatography/tandem mass spectrometry in negative ion mode.

The effect of ionization modifiers added to the mobile phase of nanoflow liquid chromatography-tandem mass spectrometry (nLC-ESI-MS³) on the simultaneous analysis of all phospholipid (PL) classes in negative ion mode has been investigated. While MS analysis of most PL classes is carried out in negative ion mode, analysis of neutral polar (polar but electrically neutral) lipids like phosphatidylcholine (PC) and sphingomyelin (SM) is highly efficient in positive ion mode. Therefore, analysis of PL mixture samples often requires two separate runs in both positive and negative ion mode. In order to establish run conditions to carry out a single nLC-ESI-MS-MS for all PLs, the ionization efficiency of 13 different types of PL molecules in nLC-ESI-MS has been evaluated in negative ion mode by varying the modifiers and their concentrations. Experiments demonstrated that a mixture of 0.05% ammonium hydroxide and 1 mM ammonium formate added to the mobile phase provided effective ionization for all classes of PLs. The optimized conditions were applied to the analysis of a phospholipid mixture extracted from a human urine sample, yielding the identification of a total of 85 PL species. Analysis of the same sample with dual nLC-ESI-MS² runs in both positive and negative ion mode confirmed that nLC-ESI-MS³ with the mixed modifier run only in negative ion mode gave comparable results.

A hyphenated microLC-Q-TOF-MS platform for exosomal lipidomics investigations: application to RCC urinary exosomes.

Urinary exosomes are released from every renal epithelial cell type facing the urinary space and therefore, they may carry molecular markers of renal dysfunction and structural injury. Here, we present a hyphenated microLC-Q-TOF-MS platform for lipidomics studies applied to investigate the urinary exosome lipid repertoire. Lipids were separated by reversed-phase chromatography using a linear gradient of formic acid 0.2% and tetrahydrofuran, in 40 min of analysis. Features (m/z with associated own retention time) were extracted by MarkerLynx(TM) (Waters) and processed, demonstrating good analytical performance in terms of repeatability and mass accuracy of the microLC Q-TOF MS platform. In particular, a stable retention time (RSD less than 4%) and relative intensity (RSD from 2.9% to 11%) were observed. Moreover, the method takes advantages by the use of a lock spray interface (Waters) that allows readjusting the m/z data after acquisition, obtaining inaccuracy below 6 ppm in measuring the m/z value of the reference compound during chromatographic run. The method was employed in a preliminary application to perform comparative analysis from healthy control subjects and renal cell carcinoma (RCC) patients, in order to possibly highlight differences in lipid composition to be exploited as potential tumor biomarker. Differential lipid composition in RCC urinary exosomes was achieved and tentatively identified by accurate mass, providing a preliminary indication of a relationship between lipid composition of urinary exosomes and RCC disease. Among the total features significantly different in RCC exosomes, the ion at m/z 502.3 was taken as an example for molecular confirmation by MS/MS fragmentation analysis.

Comparison of the diagnostic accuracy of di-22:6-bis(monoacylglycerol)phosphate and other urinary phospholipids for drug-induced phospholipidosis or tissue injury in the rat.

Cationic amphiphilic drugs and aminoglycoside antibiotics can induce phospholipidosis (PLD), an abnormal accumulation of phospholipids in lysosome-derived vesicles, in preclinical studies. The incidence of PLD in patients and its clinical relevance are difficult to assess without noninvasive biomarkers. Di-docosahexaenoyl bis(monoacylglycerol)phosphate (di-22:6-BMP) is a phospholipid that is enriched in lysosomal membranes and a proposed urinary biomarker of drug-induced PLD. The specificity of di-22:6-BMP for PLD was compared to other phospholipid species that can increase in urine with nephrotoxicity. Using liquid chromatography coupled to mass spectrometry, 12 phospholipids were assayed in the urine of rats treated with drugs that induced PLD or caused renal or skeletal muscle injury. In receiver operating curve analyses, urinary di-22:6-BMP was a significantly better predictor of PLD and the least predictive of tissue injury of the phospholipids assayed. The data provide evidence supporting the use of di-22:6-BMP as a urinary biomarker of PLD in rats.

Analysis of phospholipids using an open-tubular capillary column with a monolithic layer of molecularly imprinted polymer in capillary electrochromatography-electrospray ionization-tandem mass spectrometry.

In this study, an open-tubular capillary electrochromatography (OT-CEC) column with a monolithic layer of molecularly imprinted polymer (MIP) based on methacrylic acid, ethylene glycol dimethacrylate, and 4-styrenesulfonic acid was utilized for the simultaneous separation and characterization of phospholipid (PL) molecular structures by interfacing with electrospray ionization-tandem mass spectrometry (ESI-MS-MS). Introducing an MIP-based monolith along with charged species at the OT column made it possible to separate PL molecules based on differences in head groups and acyl chain lengths in CEC. For the interface of OT-CEC with ESI-MS-MS, a simple nanospray interface utilizing a sheath flow was developed and the resulting OT-CEC-ESI-MS-MS was able to separate PL standards (phosphatidylserines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acid, and lysophosphatidylglycerols). The developed method was applied to human urinary lipid extracts, and resulted in the separation and structural identification of 18 molecules by data-dependent collision-induced dissociation.

Shotgun lipidomics for candidate biomarkers of urinary phospholipids in prostate cancer.

Qualitative and quantitative profiling of six different categories of urinary phospholipids (PLs) from patients with prostate cancer was performed to develop an analytical method for the discovery of candidate biomarkers by shotgun lipidomics method. Using nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry, we identified the molecular structures of a total of 70 PL molecules (21 phosphatidylcholines (PCs), 11 phosphatidylethanolamines (PEs), 17 phosphatidylserines (PSs), 11 phosphatidylinositols (PIs), seven phosphatidic acids, and three phosphatidylglycerols) from urine samples of healthy controls and prostate cancer patients by data-dependent collision-induced dissociation. Identified molecules were quantitatively examined by comparing the MS peak areas. From statistical analyses, one PC, one PE, six PSs, and two PIs among the PL species showed significant differences between controls and cancer patients (p < 0.05, Student's t test), with concentration changes of more than threefold. Cluster analysis of both control and patient groups showed that 18:0/18:1-PS and 16:0/22:6-PS were 99% similar in upregulation and that the two PSs (18:1/18:0, 18:0/20:5) with two PIs (18:0/18:1 and 16:1/20:2) showed similar (>95%) downregulation. The total amount of each PL group was compared among prostate cancer patients according to the Gleason scale as larger or smaller than 6. It proposes that the current study can be utilized to sort out possible diagnostic biomarkers of prostate cancer.

Quantitative analysis of urinary phospholipids found in patients with breast cancer by nanoflow liquid chromatography-tandem mass spectrometry: II. Negative ion mode analysis of four phospholipid classes.

Analysis was performed on four different categories of phospholipids (phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), and phosphatidic acid (PA)) from urine in patients with breast cancer. This quantitative analysis was conducted using nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS-MS). This study shows the profiling of the phospholipids (PLs) that can be identified by the negative ion mode of MS. A previous study (Kim et al. Anal. Bioanal. Chem. 393:1649, 21) focused on only two PL classes: phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) and were identified by positive ion mode. PLs were extracted by lyophilization of 1 mL of urine from both healthy normal females and breast cancer patients before and after surgery. Separation of PLs was performed by nLC followed by structural identification of PLs using data-dependent collision-induced dissociation. A total of 34 urinary PL molecules (12 PSs, 12 PIs, four PGs, and six PAs) were quantitatively examined. Among the four PL categories examined in this study, most PL classes showed an increase in the total amounts in the cancer patients, yet PIs exhibited some decreases. The present study suggests that the lipid composition found in the urine of breast cancer patients can be utilized for the possible development of disease markers, when the analysis is performed with negative ion mode of nLC-ESI-MS-MS.

Assessing low-dose gentamicin-induced kidney injury in rats by analysis of urine.

INTRODUCTION: Gentamicin is an important aminoglycoside antibiotic used in clinics to combat infections from especially gram negative bacteria. A frequent side-effect of aminoglycoside antibiotics is a kidney injury consisting of necrosis of proximal tubular cells. It is important both in clinics and in research directed to eliminate or ameliorate this side-effect that a method is available for detection of injury at an early stage. METHODS: We have therefore compared the sensitivity of four methods currently used in animal models to assess kidney injury induced by aminoglycoside antibiotics by applying them to the analysis of urine from male rats treated with low doses of gentamicin. RESULTS: Excretion of phospholipids was significantly increased in contrast to excretion of N-acetyl-beta-glucosaminidase (NAG), neutrophil gelatinase-associated lipocalin (NGAL) and protein. Assessment of phospholipiduria thus is the more sensitive noninvasive way to monitor initial renal injury induced by aminoglycoside antibiotics. A protocol is given for the serial analysis of urinary phospholipids allowing a considerable number of determinations to be carried out in the course of 2-3 days. DISCUSSION: It is well known that all four considered methods do detect kidney damage induced in rats by high gentamicin doses far above doses used in clinics. The present investigation shows that only the analysis of the urinary phospholipids will detect damage induced by low doses of gentamicin. The method is relevant for animal model studies but will require considerable and innovative development for use in clinics.

Profiling of human urinary phospholipids by nanoflow liquid chromatography/tandem mass spectrometry.

Nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nanoLC-ESI-MS-MS) was used for the first time in a comprehensive analysis of human urinary phospholipids (PL). PL mixtures from human urine were separated with a reversed phase LC capillary column coupled to ESI-MS-MS. This study used the dual scan method in which two consecutive LC-ESI-MS-MS runs were done in both positive ion mode to detect phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and in negative ion mode to detect phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), and phosphatidylglycerol (PG). We focused on identifying the maximum number of PLs from a healthy human urine sample by varying the extracted volume of urine along with the evaluation of extraction efficiency for urinary PLs. We found that 22 PCs, 14 PEs, 15 PIs, 13 PSs, 7 PAs, and 4 PGs were identified during nLC-ESI-MS-MS when phospholipids in urine were extracted by ultracentrifugation. The efficiency of lipid extraction by ultracentrifugation versus lyophilization was evaluated by reducing the initial urine volume. We found that lyophilization was more efficient than ultracentrifugation for extracting lipids from small volumes (1 mL) of urine.

Urinary metabolic fingerprinting for amiodarone-induced phospholipidosis in rats using FT-ICR MS.

In the research and development for new therapeutic compounds, there has been a focus on detecting the changes of metabolites induced by drug administration and finding surrogate markers to assess its toxicity. We examined the suitability of urinary metabolic fingerprinting using Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) for toxicological assessment in the amiodarone (AMD)-induced phospholipidosis (PLD) rat model. There were more than 400 different ion peaks detected in the negative ion mode analysis with FT-ICR MS. About 20% of the detected ions were altered more than 1.5 fold by AMD-treatment. On the scores plot of principal component analysis (PCA), the ion profiles of the treated were separated time-dependently. The loading plot revealed that the metabolites causing PCA results were m/z 178.05101, 191.01979, 192.06676, 212.00239, 258.9944 and 283.0820. The ion at m/z 178.05101 is considered to be hippurate (HA), 192.06676 is phenylacetylglycine (PAG) and 212.00239 is indican (IDN). These results indicate that PAG, IDN and HA are biomarkers for AMD-induced PLD in urinary metabolic fingerprinting using FT-ICR MS. These markers may be useful for evaluation of chemicals, which have the potential to induce PLD.