Development of liquid chromatography mass spectrometric methods for quantification of metabolites from cellular level to clinical biomarkers

Metabolites are low molecular weight compounds participating in different functions of cellular systems. Metabolites can be used as diagnostic biomarkers for numerous diseases. Liquid chromatography tandem mass spectrometry (LC-MS/MS) is a powerful tool in quantification of metabolites from various sample matrices. Good sensitivity and specificity are the main benefits of the technique. Mass spectrometry is commonly used in industry, drug research and clinical diagnostics. Extensive validation of newly developed analytical methods will construct the basis to a reliable assay, and it is significant especially when analysing e.g. patient samples. The aim of this study was to develop quantitative assays for metabolites from biological samples for biomedical research and clinical diagnostics. We designed and constructed an on-line high performance liquid chromatography (HPLC) equipment and validated an assay for direct quantification of extracellular metabolites from cell cultivation. Automated sampling for LC-MS/MS analysis of intracellular metabolites was connected to the on-line system. The on-line analysis improves the methodology and shortens the time of analysis. Furthermore, a frequent sampling data can provide valuable information about physiological indications in various cell cultivations. On-line HPLC is suitable for various biotechnological applications because of its ability to monitor and collect data during cell cultivation. We developed and validated LC-MS/MS assays for neuroendocrine tumor (NET) biomarkers 5-hydroxyindole acetic acid (5-HIAA) and vanillylmandelic acid (VMA) from human serum. Generally, urinary HPLC assays are used for the determination of NET markers. HPLC assays have certain limitations and 24-h urine collection is laborious. Our LC-MS/MS assays are specific, fast and well suited for diagnostics of NETs. Furthermore, guidelines for urine collection advise to refrain from serotonin-containing foods for three days before sample collection. We showed that such a diet restriction before serum 5-HIAA assay is not necessary. Instead, one day serotonin-free diet before sampling is sufficient because the half-life of 5-HIAA in circulation was found to be 1.3 hours. All assays developed during this study were sensitive and had a wide linear range. Our serum 5-HIAA LC-MS/MS assay is routinely used for the analysis of NET patient samples at the Helsinki University Central Hospital Laboratory, HUSLAB. Serum VMA LC-MS/MS assay will be in routine use in the HUSLAB in near future. Furthermore, On-line HPLC Ltd, (Helsinki, Finland) has commercialized the on-line HPLC equipment developed in this study.Metaboliitit ovat pienen molekyylipainon omaavia yhdisteitä, jotka osallistuvat erilaisiin toimintoihin solujen aineenvaihdunnassa. Eri metaboliitit toimivat myös useiden sairauksien merkkiaineina ja niiden pitoisuuden mittausta käytetään apuna sairauksien toteamisessa sekä seurannassa. Nestekromatografia (LC) yhdistettynä massaspektrometriaan (MS) on tehokas analyysitekniikka metaboliittien määritykseen. LC-MS -menetelmiä käytetään laajalti esimerkiksi teollisuudessa, lääketutkimuksessa sekä kliinisessä diagnostiikassa. Menetelmän validointi eli toimivuuden testaus on erittäin tärkeä vaihe uusien määritysmenetelmien kehityksessä. Validointi takaa, että menetelmä toimii halutulla tavalla ja että analyysitulokset ovat luotettavia, mikä on erityisen tärkeää varsinkin analysoitaessa potilasnäytteitä. Tämän työn tarkoituksena oli kehittää ja validoida kvantitatiivisia määritysmenetelmiä metaboliiteille käyttäen hyödyksi LC-MS -tekniikoita. Kyseisiä menetelmiä voidaan hyödyntää biolääketieteellisessä tutkimuksessa ja kliinisessä diagnostiikassa. Suunnittelimme ja valmistimme korkean erotuskyvyn nestekromatografia (HPLC) -laitteiston ja validoimme menetelmän solunulkoisten metaboliittien reaaliaikaiseen määritykseen suoraan solukasvatuksesta. Lisäksi keräsimme näytteitä solukasvatuksesta automaattisella näytteenottolaitteistolla ja määritimme kerätyistä näytteistä solun sisäisiä metaboliitteja LC-MS/MS -menetelmällä. Reaaliaikaisen mittauksen avulla saatiin lisää hyödyllistä tietoa solujen aineenvaihdunnasta. Metaboliitit 5-hydroksyyli-indoliasetaatti (5-HIAA) ja metoksihydroksimandelaatti (MOMA, VMA) ovat neuroendokriinisten kasvaimien merkkiaineita. Yleensä näitä merkkiaineita on määritetty potilaan vuorokausivirtsasta HPLC -menetelmällä yhdistettynä elektrokemialliseen tai flurometriseen detektioon. Nämä menetelmät ovat kuitenkin alttiita häiritseville yhdisteille, kuten lääkeaineille tai toisille metaboliiteille. Lisäksi vuorokausivirtsan keräys ja esikäsittely on hankalaa ja aikaa vievää sekä potilaalle että laboratoriolle. Näiden syiden vuoksi kehitimme ja validoimme suoraviivaiset LC-MS/MS -menetelmät 5-HIAA:n ja VMA:n nopeampaan ja luotettavampaan määritykseen verinäytteestä. Kehitetyt menetelmät ovat spesifisiä, nopeita ja sopivat hyvin kliiniseen diagnostiikkaan. Aiemmat potilasohjeet neuvoivat välttämään tiettyjä ruoka-aineita kolme päivää ennen 5-HIAA virtsanäytteenottoa. Me osoitimme, että yksi päivä riittää kyseisten ruoka-aineiden välttämiseen ennen verinäytteenottoa. Kaikki tässä työssä kehitetyt määritysmenetelmät olivat herkkiä, nopeita sekä paransivat analytiikkaa. Seerumin 5-HIAA -menetelmä on ollut rutiinikäytössä Helsingin yliopistollisen keskussairaalan laboratoriossa, HUSLAB:ssa vuodesta 2013 ja seerumin VMA -menetelmä otetaan käyttöön lähiaikoina. Lisäksi On-line HPLC -niminen yritys on kaupallistanut tässä työssä kehitetyn HPLC-laitteiston

Label-free mass spectrometry proteome quantification of human embryonic kidney cells following 24 hours of sialic acid overproduction

Peer reviewe

Plasma bradykinin concentrations during septic shock determined by a novel LC-MS/MS assay

Background: Bradykinin is an important mediator of inflammation and vascular permeability and could have an important role in the development of septic shock. Measurement of bradykinin by immunological methods may suffer from interference and lack of specificity. We developed and validated a liquid chromatography mass spectrometry assay (LC-MS/MS) for plasma bradykinin. Methods: We used plasma samples from healthy volunteers (n = 19) and patients with septic shock (n = 47). Stable isotope bradykinin internal standard was added to samples before solid-phase extraction and quantification by LC-MS/MS. Stability of bradykinin was studied for 12 months. Results: Our assay has good sensitivity (0.1 nmol/l) and a wide linear range (0.1-1000 nmol/1). Bradykinin added to plasma was stable for 12 months at -20 degrees C when a mixture of protease inhibitors was added at sampling but degraded during repeated freezing and thawing. Bradykinin concentration in plasma from septic shock patients (<0.1-0.6 nmol/l) did not change significantly during shock and recovery but differed slightly from that in healthy individuals (0.5-1.1 nmol/1). Conclusions: Our bradykinin assay was successfully used to determine bradykinin concentrations in plasma samples. Intensive care unit patients with septic shock had low concentrations of plasma bradykinin during both shock and recovery phases.Peer reviewe

Comparison of serum serotonin and serum 5-HIAA LC-MS/MS assays in the diagnosis of serotonin producing neuroendocrine neoplasms : A pilot study

Background: Serotonin (5-hydroxytyramine) is a mediator of gastrointestinal smooth muscle contraction, and is secreted by neuroendocrine neoplasms (NENs). We developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for serum serotonin to be used in NEN diagnostics and follow-up. Methods: We used serum samples from healthy volunteers (n = 31) and patients suspected or monitored for NEN (n = 98). Serotonin-D-4 internal standard was added to samples before solid phase extraction (SPE) and quantification by LC-MS/MS. The effects of sample handling and preparation on serotonin stability were studied. Finally, we established a provisional reference range for serum serotonin and compared our assay with serum 5hydroxyindoleacetic acid (5-HIAA) for detection of NENs. Results: Our assay is sensitive and has a wide linear range (10-10,000 nmo1/1). Serum serotonin is stable for 7 days at room temperature and for 3 months at -20 degrees C. Sampling temperature is not critical. Normal range for serum serotonin was 270-1490 nmo1/1. We found that serum serotonin and 5-HIAA performed equally well as diagnostic tests for NENs. Conclusions: Our LC-MS/MS assay for serum serotonin is well suited for clinical research and patient diagnostics. Our results confirm that it can complement 5-HIAA in diagnosis of NENs.Peer reviewe

UriSed 3 PRO automated microscope in screening bacteriuria at region-wide laboratory organization

Background and aims: We assessed the possibility to rule out negative urine cultures by counting with UriSed 3 PRO (77 Elektmnika, Hungary) at Helsinki and Uusimaa Hospital District. Materials and methods: Bacteria counting of the UriSed 3 PRO automated microscope was verified with reference phase contrast microscopy against growth in culture. After acceptance into routine, results of bacteria and leukocyte counting from 56 426 specimens with eight UriSed 3 PRO instruments were compared against results from parallel samples cultured on chromogenic agar. Laboratory data including preanalytical details were accessed through the regional database of the Helsinki and Uusimaa Hospital District. Results: A combined sensitivity of 87-92% and a negative predictive value of 90-96% with a specificity of 54-50% was reached, depending on criteria. Preanalytical data (incubation time in bladder) combined with the way of urine collection would improve these figures if reliable. Conclusions: Complex patient populations, regional logistics and data interfases, and economics related to increased costs of additional particle counts against costs of screening cultures of all samples, did not support adaptation of a screening process of urine cultures. This conclusion was made locally, and may not be valid elsewhere.Peer reviewe

Verification of UriSed 3 PRO automated urine microscope in regional laboratory environment

Background and aims: Ten UriSed 3 PRO automated microscopes (77 Elektronika, Hungary) were verified for nine HUSLAB laboratories with 160 000 annual urine samples. Materials and methods: Particle counting of the primary UriSed 3 PRO instrument (77 Elektronika, Hungary) was verified against reference visual microscopy with 463 urine specimens, and against urine culture on chromogenic agar plates with parallel 396 specimens. Nine secondary instruments were compared pairwise with the primary instrument. Results: Relative imprecisions compared to Poisson distribution, R(CV), were estimated to be 1.0 for white blood cell (WBC) and 1.5 for red blood cell (RBC) counts, respectively. Spearman's correlations against visual microscopy were rS = 0.94 for WBC, rS = 0.87 for RBC, and rS = 0.82 for squamous epithelial cell (SEC) counts. Agreement with visual microscopy (Cohen's weighted kappa) was 0.94 for WBC, 0.89 for RBC, 0.88 for SEC, 0.59 for combined casts, and 0.49 for non-squamous epithelial cells (NEC). Bacteria were detected with a sensitivity of 90% and specificity of 39 against culture at 107 CFB/L (104 CFU/mL). Created flagging limits allowed automated reporting for 70-75% of patient results. Conclusions: UriSed 3 PRO instruments were adopted into routine use after acceptance of the verification.Peer reviewe