metabo-guideseparations.co.za/wp-content/uploads/2016/10/metabo-guide.pdf · thomas hengstl 1,2,3,...

1

Heart failure?Cheng et al. 2015,

JACCAlzheimer‘s?

Mapstone et al. 2014, Nature Medicine

Diabetes?Geurts et al. 2015,

Nature Communications

Metabo-GuideMetabolomics based biomarker

discovery

Cancer?Belcheva et al. 2014,

Cell

35011 V.1.0 2016-04-21

2

Biocrates helps researchers to identify metabolic biomarkers to predict diseases or evaluate treatment efficacy. Within the past three years, researchers have used our technology to develop biomarkers for Alzheimer’s, heart failure, breast cancer and many other diseases. More than 20 publications in the highest ranked journals like Science, Nature, Cell and over 500 publications in total proof the usefulness of our work and the readiness of the technology.

Biocrates in a nutshell

Plasma phospholipids identify anteced-ent memory impairment in older adults (Mapstone et al., 2014 in Nature Medicine)

Mapstone et al. discovered a metabolic sig-nature consisting out of 10 lipids that was able to predict the phenoconversion to AD or MCI within a 2-3 years timeframe.

Davis et al. took a closer look at the differ-ences of the human metabolome during sleep and wake cycles and discovered 27 significantly increased metabolites.

Identifiying patients who benefit from a cer-tain drug treatment is an important clinical application. Metabolomics can help to de-velop signatures to seperate responders from non-responders.

In 2015 Cheng et al. reported two panels of metabolites. The first one showed a similar diagnostic value to BNP and the second one showed a significant benefit in the prognosis of heart failure.

Increased efficacy of omalizumab (Xolar®, Novar-tis) in atopic dermatitis patients with wild-type filaggrin status and higher serum levels of phos-phatidylcholines (Hotze et al., 2014 in Allergy)

Alzheimer‘s disease Heart failure

Treatment prediction (stratification)

Effect of sleep deprivation on the human metabolome (Davies et al., 2014 in PNAS)

Metabolic disturbances identified in plasma are associated with outcomes in patients with heart failure: diagnostic and prognostic value of metabolomics (Cheng et al., 2015 in JACC)

The human metabolome during sleep

Introduction

3

ContributorsThomas Hengstl 1,2,3, Brigitte Pfurtscheller 2, Markus Langsdorf 3, Stefan Ledinger 3, Manuel Kratzke 3

1 Idea, 2 Graphics, 3 Content

Table of contentIntroductionBiocrates in a nutshell.......................................................................................................................................................................................2Table of content ..................................................................................................................................................................................................3Company Profile .................................................................................................................................................................................................4From health to disease .....................................................................................................................................................................................5The metabolome of the cell ............................................................................................................................................................................6From cell to system ............................................................................................................................................................................................7From drop to data ..............................................................................................................................................................................................8

PublicationsSuccessfully published studies ......................................................................................................................................................................9

Project GuideMetabolomics project guide ....................................................................................................................................................................... 10Metabolite menu ..............................................................................................................................................................................................11Sample types and species ............................................................................................................................................................................ 12Sample processing literature ....................................................................................................................................................................... 13

Focus on technologyWorld wide data comparability .................................................................................................................................................................. 14Systemic metabolites as biomarkers ........................................................................................................................................................ 16

AppendixCompatibility .....................................................................................................................................................................................................17Metabolite menu ............................................................................................................................................................................................. 18AbsoluteIDQ® p180 Kit Requirements ...................................................................................................................................................... 28Biocrates® Bile Acids Kit Requirements .................................................................................................................................................... 30AbsoluteIDQ® Stero17 Kit Requirements................................................................................................................................................. 32Product Portfolio ............................................................................................................................................................................................. 34

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Company Profile

BIOCRATES Life Sciences AG – The Deep Phenotyping Company

BIOCRATES was founded in Innsbruck in 2002 by three leading university professors (Günther Bonn, Adelbert Roscher, Hartmut Glossmann) from Munich and Innsbruck as well as Bionorica, a German phytocompany. BIOCRATES offers me-tabolomics solutions for targeted, quantitative and quality-controlled Metabolic Phenotyping (“Deep Phenotyping”). In 2016 Biocrates with its 45 employees ships products to almost every country world wide and has a seperate office in Washington DC to serve the US market.

Technology Targeted Metabolic Phenotyping provides the individual metabolic signature as a surrogate marker of individual genetic disposition, somatic changes, acquired adaptations and exposition to pathogens, environment and alimentation.The individual metabolic signature is a holistic system diagnosis and can be highly specific for disease diagnosis, disease sub-classification, treatment prediction, drug response and identification of pathophysiological processes underlying the different diseases. The metabolic phenotype offers the possibility to make an early diagnosis and prognosis of diseases for which a precise diagnosis on scientific medical parameters was not available, yet.The metabolite analysis is based on liquid chromatographic and mass spectrometric measurements (LC-MS). The possibility of measuring different biological matrices of different species in low sample volumes allows translational research as well as longitudinal studies.

Ready to use Kits The mass spectrometry based Kits permit precise quantification of hundreds of endogenous metabolites of different classes, including acylcarnitines, amino acids, biogenic amines, sugars, lipids, bile acids, steroids and many more.

Metabolic Phenotyping Services (Contract Research)Individualized lab services offer mass spectrometry-based quantification of more than 630 endogenous metabolites in various sample types from a wide range of biological species.

Areas of ApplicationMetabolic Phenotyping data allow detailed metabolic pathway analysis and identification of potential biomarkers or drug targets as well as their mode of action. Additionally, these results provide targets for individual therapy and disease predic-tion. In combination with other “omics”-techniques Metabolic Phenotyping supports the identification of gene functions and provides a complete system biological diagnosis for an improved personalized medicine approach.

BIOCRATES Life Sciences AGEduard-Bodem-Gasse 8 tel: +43 (0) 512 579 823 [email protected] Innsbruck. Austria fax: +43 (0) 512 579 823 - 329 www.biocrates.com

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It is generally accepted that the transition from health to disease is always connected to physiological changes. In the past few decades researches heavily developed and investigated the -”omics” disciplines to further characterize what these changes are, using a multitude of different technologies. While Genomics, Transcriptomics and Proteomics are already well appreciated technologies, Metabolomics is just about to start to make its way into the prediction, diagnosis and prognosis of diseases.

From health to disease

Health

Change

Disease

Type: Genome

Complexity: ≈2x104

Technology: Next Generation Sequencing (NGS)

Company: e.g. Illumina®, Thermo®, Pacific Biosciences®

Disease causing effect: Mutations, deletions, duplications,…

Type: Transcriptome

Complexity: ≈105

Technology: RNA-seq, Microar-ray, qPCR

Company: e.g. Illumina®, Ther-mo®, Agilent®, Qiagen®

Disease causing effect: Dysregulation of gene expression

Type: Proteome

Complexity: ≈106

Technology: Miroarray, MS/MS

Company: e.g. Affymetrix®, Ar-rayit®, Bruker, Thermo

Disease causing effect: Dysregulation of protein synthesis or degradation

Type: Metabolome

Complexity: ≈104-5

Technology: LC-MS, GC-MS, NMR

Company: e.g. Biocrates®, Waters®, Sciex, Thermo, Bruker, Metabolon

Disease causing effect: Metabolic concentration differ-ences due to dysregulation of enzyme activities

TranscriptomicsGenomics

ProteomicsMetabolomics

6

Amino acids

Urea-cycle, activity of gluconeogenesis and glycolysis, insulin sensitivity, neu-rotransmitter metabolism, oxidative stress

(Acyl)Carnitines

Energy metabolism, fatty acid transport and mitochondrial fatty acid oxidation, ketosis, oxidative stress, mitochondrial membrane damage

Sphingolipids

Signaling cascades, membrane dam-age

Bile acids

Signaling molecules, hormone-like func-tions, liver injury, regulation of drug efficacy / toxicity via CYP450

Steroid hormones

Carbohydrate / lipid metabolism, water / mineral household, reproduction process, stress

Neurotransmitters

Neurotransmitter metabolism

H

HH

H

H

H

HH

HO

O

O O

O

H

HHN

O

O

H

N

O O

O

+

-HH

HH

H

HHH

N+

N

PO

O-O

O

O

O O

H

H H

HH

O

O

H

H

H

H

O

O

N

Cholic acid Testosterone

SM-C14

Dopamine

Alanine Carnitine

The Cell is the smallest entity of the human body. It has to perform an almost unlimited number of tasks including DNA replication, RNA and protein synthesis, producing energy and maintaining the cell membrane. All these pro-cesses require energy and building blocks such as nucle-

otides, amino acids, lipids and carbohydrates which are provided by a complex system of biochemical pathways and processes. The constant synthesis and degradation of these substances is called metabolism.

The metabolome of the cellTo date over 4000 metabolites (

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Metabolic and cellular differentiation Systemic blood stream

From cell to systemThe rationale behind systemic metabolomics

Combined, sys-temic metabolic

signature

As Cells differentiate into different subtypes, also their metabolism adapts to fulfill the function of the organ, re-sulting in a specific metabolic profile. Organs are function-al entities consisting of a large number of cells perform a similar task. Since every organ is connected to the Blood stream, blood (or other fluids that are in contact with an

organ) can be used to measure and monitor metabolic signatures. It has been shown that already in a very early state, diseases can cause disturbances in these metabol-ic profiles, allowing prediction and diagnosis often much sooner than other biomarkers.

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Conducting a Metabolomics study is, compared to other -omics studies, fast, easy and cost efficient. E.g. sample collec-tion can be performed easily with dried blood spots; furthermore, processing of up to 80 patient samples can be achieved within 48 hours. Sample volumes are low; from 10 µl of blood plasma one can quantitatively measure up to 180 metabo-lites. Considering that each of them could be a disease biomarker, metabolomics has the potential of becoming a strong companion. Even if mass spectrometry experience isn’t available, outsourcing is simple, affordable and straight forward.

4 h

over night

From drop to dataHow to conduct a metabolomic study

DiseaseControl

Sample collection

Sample processing

Sample measurement

Data analysis & interpretation

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Indication No.* Successfully published studies seleced by indication

Neuroscience 21 Plasma phospholipids identify antecedent memory impairment in older adults (Mapstone et al. 2014)

Cardiovascular Diseases 15 Metabolic disturbances identified in plasma are associated with outcomes in patients with heart failure: diagnostic and prognostic value of metabolomics (Cheng et al. 2015)

Diabetes 59 Effects of metformin on metabolite profiles and LDL cholesterol in patients with type 2 diabetes (Xu et al. 2015)

Oncology 16 Alteration of amino acid and biogenic amine metabolism in hepatobiliary cancers: Find-ings from a prospective cohort study (Stepien et al. 2015)

Epidemiology & Genetics 55 A genome-wide perspective of genetic variation in human metabolism (Illig et al. 2010)

Inflammation & Immunology 26

A Conserved Circular Network of Coregulated Lipids Modulates Innate Immune Responses (Köberlin et al. 2015)

Nutrition & Lifestyle 60 Metabolic profiles of male meat eaters, fish eaters, vegetarians, and vegans from the EPIC-Oxford cohort (Schmidt et al. 2015)

Pharmacology/Toxicology 18 Increased efficacy of omalizumab in atopic dermatitis patients with wild-type filaggrin status and higher serum levels of phosphatidylcholines (Hotze et al. 2014)

Successfully published studiesRapidly growing impact in basic and clinical research

Within the past few years metabolomics has supported research accross many different fields of indications. This illustration and the table below provide an overview to selected, published studies in which Biocrates technology was used. A more complete list of papers can be found in our publication list.

* Number of published studies since 2010

Publications

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In case you choose to conduct the study on your own we provide you our software “MetIDQ” with the Kit. MetIDQ allows you to manage your projects, samples and mea-surements and offers integrated support for the whole Kit workflow. After the sample measurement, the data is imported to MetIDQ. The technical validation will then as-sign quality scores to your data, providing you detailed information on the performance of sample handling and measurement. You can then choose to perform statisti-cal analysis within MetIDQ or export your data in various formats (Microsoft Excel, CSV, XML, etc.) in order to use third party statistics software (e.g. Metaboanalyst, “R”).

In case that you decide to send your samples to Biocrates headquarter, we will perform the measurements, quality analysis and data compilation for you. We also offer data analysis starting from basic statistics to sophisticated analysis based on your needs. At the end, you will receive a detailed report from our data analysis specialists.

Metabolomics project guideStarting to get interested in metabolomics? Tell us what you would like to do and let us help you.

Sam

ple

co

llect

ion

Dat

a an

alys

isSt

udy

desi

gnSa

mpl

e pr

epar

atio

n

and

mea

sure

men

t

Sample collection is often conducted over multiple years. For metabolomic studies, we generally recommend to freeze samples (min. -80 °C) as fast as possible (e.g. within 30 min) to prevent metabolites from degradation. If you haven‘t already conducted a metabolic study, we provide sampling collection guidelines to assist you.

We are more than happy to assist you already during your planing phase with our experience. Feel free to contact us with any questions and let us help you designing your metabolomics study. Important points to consider are:

What type of samples are available? -> See sample types and species

Which metabolites should be analyzed? -> See metabolite menu

How many samples do you have?

Care free service Collaboration Kit

You haven’t done metabolomics be-fore but would like to try it.

You will receive full support from the beginning to the end. All you need to do is give us a call and we will guide you through the process. You don’t need any experience or equipment, all you need to do is send us your samples and we will deliver the data.

You have a collaborator with the ap-propriate MS equipment and MS ex-pertise.

You will send him the samples and we will send him all necessary materials to process and measure your sam-ples as well as the required software to analyze the data. An application specialist will come on-site and help with the setup of our technology.

You have done metabolomics before and you have the appropriate MS equip-ment and MS expertise in your lab.

We will send you all necessary materials to process and measure your samples as well as the required software to process and evaluate the data. An application specialist will come on-site and help with the setup of our technology.

Days, Months, Years

Mass spectrometry and metabolomics experience / equipment

MetIDQTM

Example for a clincal study design

Project Guide

11

Metabolite menuThe choice is yours. Biocrates offers the technology to detect and quantify more than 600 metabolites

Sphingolipids (15)

Signaling cascades, membrane damage

HH

HH

H

HHH

N+

N

PO

O-O

O

O

O O

SM-C14

Monosaccharides (1)

Glycolysis, oncometabolite

H

H

H

H

HO

OO

O O

O

Glucose

Vitamins (16)

Bioprocess optimization

H

H

H

H

H

S

O

O

O

N

N

Biotin

Fatty acids (31)

Fatty acid composition and metabolism

H

O

O

Lauric acid

Steroid hormones (17)


H

H H

HH

O

O

Testosterone

Amino acids (21)

Urea-cycle, activity of gluconeogenesis and glycolysis, insulin sensitivity

H

HHN

O

O

Alanine

Biogenic amines (21)

Neurotransmitter metabolism, oxidative stress, cell proliferation, apoptosis

H

HH

NO

S

OO Taurine

Oxysterols (17)

Cholesterol metabolism, oxidative stress, inflammation

HH

H HH

H

O

O

4-Beta-Hydroxycholesterol

Eicosanoids (15)

Inflammation

O

O

O

O

HH

H

H

HH

H

H

H

H

H

Prostaglandin E2

(Acyl)Carnitines (40)


H

N

O O

O

+

-

Carnitine

Glycerophospholipids (90)

Degradation of phospholipids, membrane damage, signaling cascades, fatty acid profile, dyslipidemia

H

N

O O

PO O O

OO-

+

LysoPC a 24-0

Lipids (331)

Lipid composition, cell membrane char-acterization

H

H

H

HH

N

OO

O

Ceramide_C16

Neurotransmitters (9)


H

H

H

H

O

O

N

Dopamine

Bile acids (20)

Signaling molecules, hormone-like functions, liver injury, regulation of drug efficacy / toxicity via CYP450

H

HH

H

H

H

HH

HO

O

O O

O

Cholic acid

AbsoluteIDQ p180 (188)(Kit / Service)

Our most frequently used targeted metabolomics discovery panel. 188 metabolites, which were ca-refully selected based on studies of the human blood metabolome.

Bile Acids Kit (20)(Kit / Service)

Bile acids are important indicators of the hepato-biliary function as well as the gut microbiome.

AbsoluteIDQ Stero17 (17)(Kit / Service)

Steroid hormones are responsible for the regulation of a multitude of physiological functions. (Steroid Kit is not available in USA and Canada)

Specialized assays(Service only)

Biocrates also offers a selection of assays for specific metabolic functions.

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- Monkey- Cow- Sheep- Pig- Dog

- Plasma- Serum- Tissue

Although 95% of our samples are either human, mouse or rat, we have already conducted numerous studies with samples from other species including:

The used sample material typically depends on the sci-entific question. While plasma and serum are by far the most common samples but other materials are also of high interest, such as CSF for neurodegenerative diseas-es or dried blood spots for new born screening.

Sample types and biological matrices

Species of origin

For other types of samples / matrices or species please contact us directly: [email protected]

Sample types and species

AbsoluteIDQ p180 Kit Bile Acids Kit AbsoluteIDQ Stero17 Services

Plasma Yes (recommended) Yes (recommended) Yes

Plea

se c

onta

ct

serv

ices

@bi

ocra

tes.

com

Serum Yes (recommended) Yes (recommended) Yes (recommended)

Tissue Yes Yes (Liver) Yes (Tumor)

Cells Yes Not tested No

Dried blood spots Yes Not tested No

Saliva Yes No No

CSF Yes No No

Feces Yes Yes No

Urine Yes No No

- Chicken- Horse- Rabbit- Zebrafish- Chinese hamster

- C. elegans- Soy- Pichia pastoris- E. coli

- Cells- Saliva- CSF

- Feces- Urine- Dried blood spots

Sample types anlyzed in our contract research

Most frequently seen sample species in contract research

% %

13

AbsoluteIDQ p180 Kit

Plasma1. Biocrates Blood Sampling Guide for Metabolic Phenotyping2. Biocrates p180 Kit user manual

Serum1. Biocrates Blood Sampling Guide for Metabolic Phenotyping2. Biocrates p180 Kit user manual

Tissue

1. Biocrates Tissue Sampling Guide for Metabolic Phenotyping2. Römisch-Margl, et al (2012): Procedure for tissue sample preparation and metabolite extraction for high-throughput targeted metabolomics.3. Biocrates Application Note - A High-Throughput Method for Targeted Metabolomics Analysis of Different Tissue Samples using the AbsoluteIDQ® Kit

Cells1. Biocrates SOP - Cell lysis method for MS-based metabolite analysis2. Targeted Metabolomics Analysis of Cell Culture Lysates using the AbsoluteIDQ® p180 Kit

Dried blood spots1. Biocrates SOP - Analysis of Dried Blood Spots (DBS) Using the AbsoluteIDQ® p180 Kit2. Zukunft, Sven; et al (2013): Targeted Metabolomics of Dried Blood Spot Extracts.

Saliva Dame, et al. (2015): The human saliva metabolome.

CSF Biocrates SOP - Analysis of Cerebrospinal Fluid (CSF) using the AbsoluteIDQ® p180 Kit

Feces Biocrates SOP - Analysis of human fecal samples with the AbsoluteIDQ® p180 Kit

Urine1. Biocrates SOP - Analysis of Urine Using the AbsoluteIDQ® p180 Kit on SCIEX 40002. Bouatra, et al. (2013): The human urine metabolome.

Biocrates Bile Acids Kit

Plasma1. Biocrates Blood Sampling Guide for Metabolic Phenotyping2. Biocrates Bile Acids Kit user manual

Serum1. Biocrates Blood Sampling Guide for Metabolic Phenotyping2. Biocrates Bile Acids Kit user manual

Tissue 1. Biocrates Tissue Sampling Guide for Metabolic Phenotyping2. Biocrates SOP - Analysis of Mouse Liver Tissue Extracts with Biocrates Bile Acids Kit

Feces Biocrates SOP - Analysis of human fecal samples with the Biocrates Bile Acids Kit

AbsoluteIDQ Stero17 Kit

Serum1. Biocrates Blood Sampling Guide for Metabolic Phenotyping2. Biocrates Stero17 Kit user manual

Sample processing literature

Literature:

Yu, Zhonghao et al. (2011): Differences between human plasma and serum metabolite profiles. In: PloS one 6 (7), S. e21230. DOI: 10.1371/journal.pone.0021230.

Plasma = Serum, fibrinogen & clotting factors

Serum = Fluid after blood has clotted

Erythrocytes

Plasma

Leucocytes

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World wide data comparabilityBiocrates Kits guarantee data comparability arround the world and over time

Multi-platform comparability-> With a cross-platform CV of less than 15% the Biocrates p180 fullfilles the FDA guidelines for industry.

To test the robustness of the AbsoluteIDQ® p180 Kit spiked reference plasma samples were measured. Run conditions were as followed: n = 150 replicates, 26 different Kit runs, 3 different mass spectrometry plat-forms (SCIEX, Waters, Thermo), three different operators, time period of two months.Box Whisker plot on the right with 10-90% percentiles. All metabolite class-es achieve a CV The quantification of a NIST standard yielded highly accurat concentrations across many different MS platforms.

To test the accuracy of our technology, human reference plasma (SRM 1950) from NIST (National Institute of Standards and Technology) was processed using the Biocrates p180 Kit and measured on different MS platforms.

Accuracy test using NIST

Focus on technology

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In vitro diagnostic-like performance-> Although our p180 is a RUO (research use only) kit you can expect an in vitro diagnostic (IVD) like performance.

Accuracy of the AbsoluteIDQ® p180 Kit was further tested by comparing it to an approved IVD LC-MS assay from Quest Diagnostics. Results from the Biocrates p180 are highly comparable and within the expected normal range.

Multi-center-> Succeeding in an international ring trial ensures excellent performance of the p180 arround the world.

The world-wide first ring trial with the AbsoluteIDQ® p180 Kit, was initiated by Dr. Hector Keun´s group at the Imperial College in London. In five dif-ferent laboratories: Imperial College (UK), Cambridge University (UK), In-stitute of Cancer Research (UK), Intern. Agency for Research on Cancer (France), Helmholtz Zentrum München (Germany) with 3 different MS/MS instrument platforms were invited and participated at this ring trial. The ring trial enabled the Kit analysis of a defined set of quality control materials (Biocrates QC, QC pool, NIST material) and real samples (BS1-5, TS7-10) in each lab. The NIST standard reference material (SRM) 1950 was used for data batch-to-batch normalization. In the result, excellent analytical accuracy (data not shown here) and preci-sion (inter-lab precision 7.4%) could be obtained demonstrating the excel-lent robustness and reliability of the p180 Kit across different labs. The ring trial results are planned to be published in 2016.

lipemic sample

Biocrates compared to Quest

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Systemic metabolites as biomarkersBiomarkers definitions and their use in the use in clinical environment

A biomarker is “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic intervention” (National Institutes of Health (NIH) working group; Biomarkers Definitions Working Group, 2001).

Time

Predisposition biomarkers

Prognostic biomarker

Earlybiomarkers

Latebiomarkers

Intermediate biomarkers

Nutrition SurgeryPharma

Death

Therapeutic intervention

Hea

lthy

Dea

thD

isea

se

“The best way to treat a disease is to detect ear-ly and subtle deviations from the healthy state and to prevent the clinical manifestation of the disorder, before typically irreversible damage has been done and more costly interventions are required (as explained by Ellis et al., 2007). Metabolomics has repeatedly shown to be able to de-tect such subtle changes before pathological findings occur with other methods.” (Citation and adapted il-lustration taken from Shushan B. et al. , 2010)

Figure illustrating the difference between diagnostic, prognostic and predictive biomarkers

Diagnostic biomarkers are able to separate individuals with a disease from healthy controls. E.g. Vouk H. et al., 2012, Discovery of phosphatidylcholines and sphin-gomyelins as biomarkers for ovarian endometriosis).

Prediction biomarkers are able to identify patients, who will respond to a certain treatment. E.g. HER2 positive patients will most likely respond to the Herceptin antibody. This is also called patient stratification. E.g. Hotze M. et al., 2014, Increased efficacy of omalizumab in atopic dermatitis patients with wild-type filaggrin status and higher serum levels of phosphatidylcholines.

Prognostic biomarkers are able to predict the likely course of a disease without therapeutic intervention. E.g. Cheng W. et al., 2015, Metabolic disturbances identified in plasma are associated with outcomes in patients with heart failure: diagnostic and prog-nostic value of metabolomics.

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Appendix

Compatibility

Biocrates Kits – MS Compatibility

AbsoluteIDQ® p150 Kit AbsoluteIDQ® p180 Kit AbsoluteIDQ® Stero17 Kit A) Biocrates® Bile Acids Kit

Waters Xevo TQ MS YES YES YES YES

Waters Xevo TQ-S YES YES 05-2016 YES

Waters Xevo TQ-S micro NO C) YES 05-2016 YES

Thermo TSQ Vantage YES YES YES YES

SCIEX 3200 (API/QTRAP) YES NO B) NO B) NO B)

SCIEX 4000 (API/QTRAP) YES YES YES YES

SCIEX 4500 (Triple Quad/QTRAP) YES YES NO C) NO C)

SCIEX 5500 (Triple Quad/QTRAP) YES YES YES YES

SCIEX 6500 (Triple Quad/QTRAP) NO C) 05-2016 NO C) NO C)

Biocrates Kits – LC Compatibility

All HPLC / UHPLC capable systems are compatible.

AbsoluteIDQ® p150 Kit AbsoluteIDQ® p180 Kit AbsoluteIDQ® Stero17 Kit A) Biocrates® Bile Acids Kit

Waters Xevo TQ MS

Flow

inje

ctio

n an

alys

is (F

IA) o

nly

UHPLC HPLC / UHPLC UHPLC

Waters Xevo TQ-S UHPLC UHPLC UHPLC

Waters Xevo TQ-S micro UHPLC UHPLC UHPLC

Thermo TSQ Vantage HPLC / UHPLC UHPLC UHPLC

SCIEX 3200 (API/QTRAP) NO NO NO

SCIEX 4000 (API/QTRAP) HPLC HPLC HPLC

SCIEX 4500 (Triple Quad/QTRAP) HPLC / UHPLC NO NO

SCIEX 5500 (Triple Quad/QTRAP) HPLC / UHPLC HPLC HPLC / UHPLC

SCIEX 6500 (Triple Quad/QTRAP) HPLC / UHPLC NO NO

All kits are for research use only (RUO).

A) Not available in USA and CanadaB) Sensitivity not sufficientC) Methods have not been developed

18

Metabolite menuThe choice is yours. Biocrates offers the technology to detect and quantify over 600 metabolites

We provide

the Phenotype

to the Genotype!

DocNr. 35006 V 2.0 2016-02-26

Sphingolipids (15)

Signaling cascades, membrane damage

HH

HH

H

HHH

N+

N

PO

O-O

O

O

O O

SM-C14

Monosaccharides (1)

Glycolysis, oncometabolite

H

H

H

H

HO

OO

O O

O

Glucose

Vitamins (16)

Bioprocess optimization

H

H

H

H

H

S

O

O

O

N

N

Biotin

Fatty acids (31)

Fatty acid composition and metabolism

H

O

O

Lauric acid

Steroid hormones (17)


H

H H

HH

O

O

Testosterone

Amino acids (21)

Urea-cycle, activity of gluconeogenesis and glycolysis, insulin sensitivity

H

HHN

O

O

Alanine

Biogenic amines (21)

Neurotransmitter metabolism, oxidative stress, cell proliferation, apoptosis

H

HH

NO

S

OO Taurine

Oxysterols (17)

Cholesterol metabolism, oxidative stress, inflammation

HH

H HH

H

O

O

4-Beta-Hydroxycholesterol

Eicosanoids (15)

Inflammation

O

O

O

O

HH

H

H

HH

H

H

H

H

H

Prostaglandin E2

(Acyl)Carnitines (40)


H

N

O O

O

+

-

Carnitine

Glycerophospholipids (90)

Degradation of phospholipids, membrane damage, signaling cascades, fatty acid profile, dyslipidemia

H

N

O O

PO O O

OO-

+

LysoPC a 24-0

Lipids (331)

Lipid composition, cell membrane char-acterization

H

H

H

HH

N

OO

O

Ceramide_C16

Neurotransmitters (9)


H

H

H

H

O

O

N

Dopamine

Bile acids (20)

Signaling molecules, hormone-like functions, liver injury, regulation of drug efficacy / toxicity via CYP450

H

HH

H

H

H

HH

HO

O

O O

O

Cholic acid

AbsoluteIDQ p180 (188)(Kit / Service)

Our most frequently used targeted metabolomics discovery panel. 188 metabolites, which were ca-refully selected based on studies of the human blood metabolome.

Bile Acid Kit (20)(Kit / Service)

Bile acids are important indicators of the hepato-biliary function as well as the gut microbiome.

AbsoluteIDQ Stero17 (17)(Kit / Service)

Steroid hormones are responsible for the regulation of a multitude of physiological functions. (Steroid Kit is not available in USA and Canada)

Specialized assays(Service only)

Biocrates also offers a selection of assays for specific metabolic functions.

19

List of Metabolites: Metabolic Phenotyping Kits & Services

At the BIOCRATES Metabolic Phenotyping Services Center we make our know-how available to partners in the industry and aca-demic sector. We quantify more than 600 metabolites, analyze a wide range of species and samples, and adapt methods to specific needs. Our services include metabolite pathway analysis to assist the annotation of results and biological interpretation of data.The number of metabolites indicates the total theoretical number of metabolites which are detected by the assay. Depending on the sample type and species as well as the used mass spectrometer, certain metabolites might be below the detection limit.

For Contract Research Services, please contact: [email protected] Kits and general questions, please contact: [email protected]

METABOLITE CLASSES

Number ofMETABOLITES

BIOCRATES KITSIn HouseSERVICES

Sample Volume 1)

Acylcarnitines 40



Service 1 10 µL 3)

Amino acids 21

Monosaccharides 1

Glycerophospholipids 90

Sphingolipids 15

Biogenic amines 21 –

Steroid hormones 17 Stero17 Kit 2) Service 2 250-500 µL

Bile acids 20 Bile Acids Kit Service 3 10 - 20 µL

Neurotransmitters 9 – Service 4 20 µL

Eicosanoids 15 – Service 5 30 µL

Fatty acids (total/free) 31 – Service 6a, 6b 35 µL

Lipids 331 – Service 7 30 µL

Oxysterols (free) 17 – Service 8 30 µL

Vitamin Assay 16 – Service 9 30 µL

1) Human Plasma; for other matrices please contact [email protected]

2) Steroid Kit is not available in USA and Canada

We provide

the Phenotype

to the Genotype!

20

AbsoluteIDQ® p180: Kit / Service 1

Acylcarnitines (40)C0 Carnitine C10:1 Decenoylcarnitine

C2 Acetylcarnitine C10:2 Decadienylcarnitine

C3 Propionylcarnitine C12 Dodecanoylcarnitine

C3:1 Propenoylcarnitine C12:1 Dodecenoylcarnitine

C3-OH Hydroxypropionylcarnitine C12-DC Dodecanedioylcarnitine

C4 Butyrylcarnitine C14 Tetradecanoylcarnitine

C4:1 Butenylcarnitine C14:1 Tetradecenoylcarnitine

C4-OH (C3-DC) Hydroxybutyrylcarnitine C14:1-OH Hydroxytetradecenoylcarnitine

C5 Valerylcarnitine C14:2 Tetradecadienylcarnitine

C5:1 Tiglylcarnitine C14:2-OH Hydroxytetradecadienylcarnitine

C5:1-DC Glutaconylcarnitine C16 Hexadecanoylcarnitine

C5-DC (C6-OH)Glutarylcarnitine (Hydroxyhexanoylcarnitine)

C16:1 Hexadecenoylcarnitine

C5-M-DC Methylglutarylcarnitine C16:1-OH Hydroxyhexadecenoylcarnitine

C5-OH (C3-DC-M)Hydroxyvalerylcarnitine (Methylmalonylcarnitine)

C16:2 Hexadecadienylcarnitine

C6 (C4:1-DC) Hexanoylcarnitine (Fumarylcarnitine)

C16:2-OH Hydroxyhexadecadienylcarnitine

C6:1 Hexenoylcarnitine C16-OH Hydroxyhexadecanoylcarnitine

C7-DC Pimelylcarnitine C18 Octadecanoylcarnitine

C8 Octanoylcarnitine C18:1 Octadecenoylcarnitine

C8:1 (p150 only) Octenoylcarnitine C18:1-OH Hydroxyoctadecenoylcarni-tine

C9 Nonaylcarnitine C18:2 Octadecadienylcarnitine

C10 Decanoylcarnitine

Amino Acids (21)Ala 3) Alanine Lys 3) Lysine

Arg Arginine Met Methionine

Asn 3) Asparagine Orn Ornithine

Asp 3) Aspartate Phe Phenylalanine

Cit 3) Citrulline Pro Proline

Gln Glutamine Ser Serine

Glu3) Glutamate Thr Threonine

Gly Glycine Trp Tryptophan

His Histidine Tyr Tyrosine

Ile 3) 4) Isoleucine Val Valine

Leu 3) 4) Leucine

Monosaccharides (1)Sum of Hexoses (including Glucose)

3) not in p150 Kit

4) p150 Kit: sum of Ile + Leu

Abs

olut

eID

Q® p

180:

Kit

/ Ser

vice

1

21

Glycerophospholipids (90)lysoPC a C14:0 PC aa C34:1 PC aa C42:0 PC ae C38:2

lysoPC a C16:0 PC aa C34:2 PC aa C42:1 PC ae C38:3





lysoPC a C18:2 PC aa C36:2 PC ae C30:0 PC ae C40:2






lysoPC a C28:0 PC aa C38:1 5) PC ae C34:1 PC ae C42:1


PC aa C24:0 PC aa C38:4 PC ae C34:3 PC ae C42:3




PC aa C30:2 5) PC aa C40:2 PC ae C36:3 PC ae C44:4



PC aa C32:2 PC aa C40:5 PC ae C38:0

PC aa C32:3 PC aa C40:6 PC ae C38:1

Sphingolipids (15)SM (OH) C14:1 SM C18:0 SM (OH) C22:1 SM (OH) C24:1

SM C16:0 SM C18:1 SM (OH) C22:2 SM C26:0

SM C16:1 SM C20:2 SM C24:0 SM C26:1

SM (OH) C16:1 SM C22:3 5) SM C24:1

Biogenic Amines (21)Ac-Orn Acetylornithine PEA Phenylethylamine

ADMA Asymmetric dimethylarginine cis-OH-Pro cis-4-Hydroxyprolinealpha-AAA alpha-Aminoadipic acid trans-OH-Pro trans-4-HydroxyprolineCarnosine Carnosine Putrescine Putrescine

Creatinine Creatinine Sarcosine (UHPLC only) Sarcosine

DOPA DOPA SDMA Symmetric dimethylarginine

Dopamine Dopamine Serotonin Serotonin

Histamine Histamine Spermidine Spermidine

Kynurenine Kynurenine Spermine Spermine

Met-SO Methionine sulfoxide Taurine Taurine

Nitro-Tyr 6) Nitrotyrosine

5) SCIEX only

6) SCIEX / Waters only

Abs

olut

eID

Q® p

180:

Kit

/ Ser

vice

1

22

Stero17/SteroIDQ: Kit / Service 2

Steroid Hormones (17)Aldosterone 11-Deoxycortisol

Androstenedione Dehydroepiandrosterone-sulfate (DHEA-S)

Androsterone β-Estradiol (E2)Corticosterone Estrone (E1)

Cortisol Etiocholanolone

Cortisone 17α-Hydroxyprogesterone

Dehydroepiandrosterone (DHEA) Progesterone

11-Deoxycorticosterone Testosterone

Dihydrotestosterone (DHT) (Stero17 only)

Biocrates® Bile Acids: Kit / Service 3

Bile Acids (20)CA Cholic acid

CDCA Chenodeoxycholic acid

DCA Deoxycholic acid

GCA Glycocholic acid

GCDCA Glycochenodeoxycholic acid

GDCA Glycodeoxycholic acid

GLCA Glycolithocholic acid

GUDCA Glycoursodeoxycholic acid

HDCA Hyodeoxycholic acid

LCA Lithocholic acid

MCA α Alpha-Muricholic acid

MCA β Beta-Muricholic acid MCA ω Omega-Murichoclic acid TCA Taurocholic acid

TCDCA Taurochenodeoxycholic acid

TDCA Taurodeoxycholic acid

TLCA Taurolithocholic acid

TMCA α/β Tauromuricholic acid (sum of alpha and beta) TUDCA Tauroursodeoxycholic acid

UDCA Ursodeoxycholic acid

23

Neurotransmitter Assay: Service 4

Neurotransmitters (9) γ-Amino-butyric acid (GABA) GlutamateDOPA Glutamine

Dopamine 5-Hydroxyindole-3-acetic acid (5-HIAA)

Epinephrine Serotonin

Norepinephrine

Eicosanoid Assay: Service 5

Eicosanoids and other Oxidation Products of Polyunsaturated Fatty Acids (PUFAs) (15) Leukotriene B4 9S-HODE

Thromboxane B2 13S-HODE

Leukotriene D4 14(15)-EpETE

Prostaglandin E2 12S-HETE

8-iso-Prostaglandin F2α 15S-HETE

Prostaglandin F2α Arachidonic acid

6-keto-Prostaglandin F1α Docosahexaenoic acid

Prostaglandin D2

Fatty Acids Assay: Service 6a (free) and 6b (total)

Fatty Acids (31/32)C12:0 Lauric acid C19:0 Nonadecanoic acid

C13:0 Tridecanoic acid cis-C18:3w6 γ-Linolenic acidC14:0 Myristic acid cis-C18:3w3 Linolenic acid

cis-C14:1w5 Myristoleic acid cis-C18:4w3 Stearidonic acid

C15:0 Pentadecanoic acid C20:0 Arachidic acid (Eicosanoic acid)

C16:0 Palmitic acid cis-C20:2w6 cis-11,14-Eicosenoic acid

cis-C16:1w10 Sapienic acid cis-C20:1w9 cis-11-Eicosenoic acid

cis-C16:1w7 Palmitoleic acid C21:0 Heneicosanoic acid

C17:0 Heptadecanoic acid cis-C20:3w6 cis-8,11,14-Eicosatrienoic acid

C18:0 Stearic acid cis-C20:4w6 Arachidonic acid

cis-C18:1w9 Oleic acid C22:0 Behenic acid

cis-C18:1w7 Vaccenic acid cis-C20:5w3 EPA (cis-5,8,11,14,17-Eicosapentaenoic acid)

cis-C18:2w6 Linoleic acid cis-C22:1w9 7) Erucic acid

cis-C22:4w6 Adrenic acid (cis-7,10,13,16-Docosatetra enoic acid) C23:0 Tricosanoic acid

cis-C22:5w3 DPA (cis-7,10,13,16,19-Docosapenta-enoic acid C24:0 Lignoceric acid

cis-C22:6w3 DHA (cis-4,7,10,13,16,19-Docosahexa-enoic acid) cis-C24:1w9 Nervonic acid

24

Lipid Assay: Service 7

Glycerophospholipids (162)Lysophosphatidylcholines (acyl or ether bond) PC ae C34:0 PC ae C38:1

LPC a C16:0 LPC a C18:2 PC ae C34:1 PC ae C38:2

LPC a C18:0 LPC a C20:4 PC ae C34:2 PC ae C38:3

LPC a C18:1 LPC e C18:0 PC ae C34:3 PC ae C38:4

Phosphatidylcholines (diacyl bonds) PC ae C34:6 PC ae C38:5



PC aa C30:2 PC aa C38:1 Phosphatidylserines (diacyl bonds)

PC aa C32:0 PC aa C38:2 PS aa C34:1 PS aa C36:2










PC aa C36:3 PS aa C40:3 PS aa C42:5

Lysophosphatidylglycerols (ether bond) PS aa C40:4

LPG e C14:2 Phosphatidylserines (acyl/ether bonds)

Phosphatidylglycerols (diacyl bonds) PS ae C34:2 PS ae C36:2

PG aa C30:0 PG aa C34:3 PS ae C36:1 PS ae C38:4

PG aa C32:0 PG aa C36:0 Phosphatidylglycerols (acyl/ether bonds)

PG aa C32:1 PG aa C36:1 PG ae C32:0 PG ae C34:1

PG aa C33:6 PG aa C36:2 PG ae C34:0 PG ae C36:1

PG aa C34:0 PG aa C36:3 Lysophosphatidylethanolamines (acyl or ether bond)

PG aa C34:1 PG aa C36:4 LPE a C16:0 LPE a C22:4

PG aa C34:2 PG aa C38:5 LPE a C18:0 LPE a C22:5

Phosphatidylcholines (acyl/ether bonds) LPE a C18:1 LPE a C22:6

PC ae C32:0 PC ae C36:3 LPE e C18:0

PC ae C32:1 PC ae C36:4 LPE a C20:4

PC ae C32:6 PC ae C36:5

Phosphatidylethanolamines (diacyl bonds) Phosphatidylethanolamines (acyl/ethyl bonds)

PE aa C20:0 PE aa C38:0 PE ae C34:1 PE ae C38:5





Lipi

d A

ssay

: Ser

vice

7

25








PE aa C36:3 PE aa C40:6



Sphingolipids (33)SM C3:0 Sphingomyelin with C3:0 SM C21:3 Sphingomyelin with C21:3

SM C14:0 Sphingomyelin with C14:0 SM C22:0 Sphingomyelin with C22:0















SM C21:2 Sphingomyelin with C21:2

Ceramides (136)Ceramides N-C27:0(OH)-Cer N-C28:0(OH)-Cer

N-C7:0-Cer N-C18:0-Cer 2-Hydroxyacyl-dihydroceramides

N-C7:1-Cer N-C18:1-Cer N-C7:0(OH)-Cer(2H) N-C19:0(OH)-Cer(2H)









Lipi

d A

ssay

: Ser

vice

7

26


N-C12:0-Cer N-C23:1-Cer N-C18:0(OH)-Cer(2H)

N-C12:1-Cer N-C24:0-Cer Dihydroceramides

N-C13:0-Cer N-C24:1-Cer N-C7:0-Cer(2H) N-C18:0-Cer(2H)









N-C17:1-Cer N-C11:1-Cer(2H) N-C22:1-Cer(2H)

2-Hydroxyacyl-ceramides N-C12:0-Cer(2H) N-C23:0-Cer(2H)

N-C7:0(OH)-Cer N-C17:0(OH)-Cer N-C12:1-Cer(2H) N-C23:1-Cer(2H)











N-C16:0(OH)-Cer N-C26:1(OH)-Cer

Lipi

d A

ssay

: Ser

vice

7

27

Oxysterol Assay: Service 8

Free Oxysterols (17)4ß-Hydroxycholesterol 24,25-Epoxycholesterol

7α-Hydroxycholesterol 7α-Hydroxycholestenone

7ß-Hydroxycholesterol 7-Ketocholesterol

22R-Hydroxycholesterol 7-Dehydrocholesterol

24S-Hydroxycholesterol 24,25-Dihydrolanosterol

25-Hydroxycholesterol 5α,6ß-Dihydroxycholestanol (THC)

27-Hydroxycholesterol Desmosterol

5α,6α-Epoxycholesterol Lanosterol

5ß,6ß-Epoxycholesterol

Vitamin Assay: Service 9

Vitamins (16) 8)

α-Tocopherol (E) Niacin (B3)

Betaine Nicotinamide (B3)

Biotin (B7) Pantothenic acid (B5)

Choline Pyridoxal-5’-phosphate (B6)

Cyanocobalamin (B12) Pyridoxine (B6)

δ-Tocopherol (E) Retinol (A)Folic acid (B9) Riboflavin (B2)

γ-Tocopherol (E) Thiamine (B1)

8) In cell culture medium only

28

Nitrogen evapo-rator or pressure manifold

(must be located in a fume hood!)

Nitrogen evaporator

Models: Techne, Porvair, VLM or Organomation

Pressure manifold

Models: Waters® Positive Pressure-96 Processor or Biotage® PRESSURE + 96 Manifold

Centrifuge Must be able to centrifuge 96-well plates of 5 cm height at 500 x g Not required when a pressure manifold is used

ShakerAny model with adjustable speed (450 - 1200 rpm) and a tray for 96-well plates as well as vials is sufficient.

Recommended: Eppendorf MixMate or Thermomixer

Pipettes

Eppendorf Multipipette® stream with 1.0, 2.5 and 10 mL tips or similar electronic model (important for reproducibility)

Single Channel: Volume range from 10 µL to 1000 µL

8-channel (check volume range with Biocrates customer support)

Vortexer Any model

Balance Accuracy < 1 mg

Volumetric flasks from 50 to 1000 mL

Trip

le q

uadr

upol

e M

S-sy

stem

SCIEX® Waters® Thermo Scientific

SCIEX 4000, 4500 or 5500 series with TurboV™ Ion Source

Waters Xevo® TQ MS, TQ-S or TQ-S micro with ESI source

Thermo Scientific TSQ VantageTM with HESI-II Ion Source

LC S

yste

m a

nd

Auto

sam

pler

Binary HPLC system with degasser unit and column oven

A 96-well plate autosampler with temperature control (10 °C) and with an injector capable of piercing a silicone mat. Sample Loop: 20 µL

Waters ACQUITY UPLC® System:Sample manager, solvent manager and column oven.Injection volume: 20 µL for standard system, 10 µL for I-Class or H-Class system


A 96-well plate autosampler with temperature control (10 °C) and with an injector capable of piercing a silicone mat. Injection volume: 10 µL.

Colu

mn

HPLC: Agilent Zorbax Eclipse XDB C18, 3.0 x 100 mm, 3.5 µm (AN: 961967-302)

UHPLC (only 4500 and 5500 series):Waters ACQUITY UPLC BEH C18 1.7 µm 2.1 x 75 mm (No. 186005604)

Waters ACQUITY UPLC BEH C18 1.7 µm 2.1 x 75 mm (No. 186005604)

HPLC: Thermo Scientific Hypersil GOLD 3.0 µm 2.1 x 100 mm (No.25003-102130)

UHPLC: Thermo Scientific Syncronis aQ 1.7 µm 2.1 x 50 mm (No. 97302-052130)

Pre-

Colu

mn HPLC: SecurityGuard, Phenomenex,

C18, 4 x 3 mm (AN: AJO-4287)UHPLC (only 4500 and 5500 series): Waters ACQUITY BEH C18 1.7 µm VANGUARD (No. 186003975)

Waters ACQUITY BEH C18 1.7 µm VANGUARD (No. 186003975)

HPLC: Thermo Scientific Hypersil GOLD Drop-in Guard Cartridges 3.0 x 10 mm (No. 25003-012101)UHPLC: Thermo Scientific 2.1 mm ID Replacement Filter Cartridge, 0.2 µm (No. 22180)

Pre-

Colu

mn

Hol

der

SecurityGuard, Phenomenex (AN: KJO-4282) (HPLC only)

Already included in the pre-column package

HPLC: Thermo Scientific UNIGUARD Drop-in Guard Cartridge Holder 2.1 mm ID (No. 852-00)UHPLC: Thermo Scientific UHPLC Direct Connect Filter Holder (No. 27006)

Mass Spectrometer

Laboratory Equipment

AbsoluteIDQ® p180 Kit Requirements

29

Software

Ethanol, Methanol, Acetonitrile, Water, Isopropanol HPLC grade

Formic acid LC-MS grade/puriss p.a. (for example Sigma Aldrich 56302)

Phenylisothio cyanate sequencing grade (for example Sigma Aldrich 317861)

Pyridine p.a. grade or higher

Ammonium acetate p.a. grade

Phosphate buffered saline (PBS) p.a. grade (for example Sigma Aldrich P4417)

Solvents and Chemicals

Mas

s Sp

ectr

omet

er

Soft

war

e


Analyst Software Version 1.5.2 or la-ter must be used for data acquisition. It is recommended to install Analyst also on another computer. The Biocrates MetIDQTM software (provided with the Kit) requires Analyst for data processing.

MassLynx V4.1

TargetLynx V4.1XcaliburTM 2.2 or later

Com

pute

r S y

stem

- MetIDQTM Software (provided by Biocrates)

- Windows Vista Service Pack 2 or higher (recommended: Windows 7)

- Microsoft .NET Framework 4.5.2 or higher

- Microsoft Visual C++ 2005 (32 & 64 bit), 2008 (32 & 64 bit), 2010 (32 & 64 bit), 2012 (32 & 64 bit) and 2013 (32 & 64 bit) Redistributable

30

Nitrogen evapo-rator or pressure manifold


Nitrogen evaporator

Models: Techne, Porvair, VLM or Organomation

Pressure manifold

Models: Waters® Positive Pressure-96 Processor or Biotage® PRESSURE + 96 Manifold

Centrifuge Must be able to centrifuge 96-well plates of 5 cm height at 500 x g Not required when a pressure manifold is used


Recommended: Eppendorf MixMate or Thermomixer

Pipettes

Eppendorf Multipipette® stream with 1.0 and 10 mL tips or similar electronic model (important for reproducibility)

Single Channel: Volume range from 10 µL to 1000 µL


Vortexer Any model

Balance Accuracy < 1 mg


Trip

le q

uadr

upol

e M

S-sy

stem


Triple quadrupole mass spectrometer LC MS/MS system:

SCIEX 4000 or 5500 series with TurboVTM Ion Source

Triple quadrupole mass spectrometer:


Triple quadrupole mass spectrometer:


LC S

yste

m a

nd

Auto

sam

pler


A 96-well plate autosampler with temperature control (10°C) and with an injector capable of piercing a silicone mat. Injection Volume: 10 µL (4000 series), 5 µL (5500 series)

Waters ACQUITY UPLC® System:Sample manager, solvent manager and column oven.Injection volume: 20 µL (TQ MS), 10 µL (TQ-S and TQ-S micro)

Binary UHPLC system with degasser unit and column oven

A 96-well plate autosampler with tem-perature control (10 °C) and with an injector capable of piercing a silicone mat. Injection Volume: 20 µL.

Colu

mn

HPLC / UHPLC: Biocrates Product Number 91220052120868

Pre-

Colu

mn

HPLC / UHPLC: Biocrates Product Number 91220052120875

Mass Spectrometer


Biocrates® Bile Acids Kit Requirements

31

Software

Methanol, Acetonitrile, Water, Isopropanol HPLC grade

Formic acid LC-MS grade/puriss p.a. (for example Sigma Aldrich 56302)

Ammonium acetate p.a. grade

Phosphate buffered saline (PBS) p.a. grade (for example Sigma Aldrich P4417)


Mas

s Sp

ectr

omet

er

Soft

war

e


Analyst Software Version 1.5.2 or later must be used for data acquisition. It is recommended to install Analyst also on another computer. The Biocrates MetIDQTM software (provided with the Kit) requires Analyst for data processing.

MassLynx V4.1


Com

pute

r S y

stem

- MetIDQTM Software (provided by Biocrates)

- Windows Vista Service Pack 2 or higher (recommended: Windows 7)

- Microsoft .NET Framework 4.5.2 or higher

- Microsoft Visual C++ 2005 (32 & 64 bit), 2008 (32 & 64 bit), 2010 (32 & 64 bit), 2012 (32 & 64 bit) and 2013 (32 & 64 bit) Redistributable

32

Vacuum-Based System and Nitro-gen evaporator or pressure manifold


Vacuum-based:

Nitrogen evaporator and heating block

Recommanded: Evaporatorsystem EVA-LS1-MT-S (incl. evaporator unit, metalblock-thermostat, heating block), VLM (V.830.211.105)

Vacuum pump

For example: Vacuum Pump, Waters (WAT085114, WAT085123 or WAT085115)

Vacuum manifold for 96-well SPE plates

Recommanded:

- Extraction Plate Manifold for Oasis 96-Well Plates, Waters (186001831)

or

- PlatePrep 96-well Vacuum Manifold, starter kit, Sigma-Aldrich (575650-U)

Pressure manifold:

- Waters® Positive Pressure-96 Processor, Waters (186006961)

or

- Biotage® PRESSURE+ 96 Positive Pressure Manifold, Biotage (PPM-96)

or

- Cerex® System 96 Processor, Cerex (288-0001)

Centrifuge For 96-well plates


Recommended: Eppendorf Thermomixer or MixMate

Pipettes


Recommended: Eppendorf Research Pro 50-1200 μL (4860000577) and tips (022492063)

Eppendorf Multipipette® stream with 1.0 and 10 mL tips or similar electronic model (important for

reproducibility)

Trip

le q

uadr

u-po

le M

S-sy

stem


SCIEX 4000 or 5500 series with TurboV™ Ion Source



LC S

yste

m a

nd

Auto

sam

pler


A 96-well plate autosampler with temperature control (10 °C) and with an injector capable of piercing a silicone mat. Injection Volume: 20 µL

Colu

mn

HPLC: Biocrates Product Number 9120052120301UHPLC: Biocrates Product Number 9120052120363

Pre-

Colu

mn


Setu

p Bo

x


Mass Spectrometer


AbsoluteIDQ® Stero17 Kit Requirements

33

Software

Acetonitrile HPLC grade

Isopropanol HPLC grade

Methanol HPLC grade

Water HPLC grade

Dichloromethane p.a. grade or higher


Mas

s Sp

ectr

o-m

eter

Sof

twar

e SCIEX® Waters® Thermo Scientific

Analyst Software Version 1.5.2 or laterMassLynx V4.1


Vortexer Any model

Ultrasonic bath Any model

Fumehood Any model


34

Product Portfolio

Accurate and quick quantifycation of metabolites in the comfort of your own lab

AbsoluteIDQ® p180 Kit

Biocrates® Bile Acids Kit

AbsoluteIDQ® Stero17 Kit

AbsoluteIDQ® Steroid Reagents

The LC-MS/MS based AbsoluteIDQ® p180 Kit is an easy-to-use research as-say for quantifying up to 186 endogenous metabolites from 5 different compound classes (i.e. acylcarnitines, amino acids, hexoses, phospho- and sphingolipids and biogenic amines). The assay requires very small sample amounts (10 μL) and shows excellent reproducibility.

Standardized analysis of bile acids from only 10 µL of either human plasma/serum (16 bile acids) or mouse plasma samples (19 bile acids).Bile Acids Phenotyping in mouse and man: • From only 10 µL of human plasma/serum or mouse plasma • 19 mouse specific bile acids • 16 human specific bile acids

AbsoluteIDQ® Stero17 Kit offers a unique experience in standardized, quantitative steroid hormone detection by simultanously analysing (multiplexing) 17 different steroid hormones with (Ultra) High Performance Liquid Chromatrography tandem mass spectrometry ((U)HPLC-MS/MS). This enables the generation of a compre-hensive steroid profile from one single human blood (serum, plasma) sample by covering a broad range of potential indications.

MS-based steroid hormone analysis has become customizable: scientists are free to use their method of choice as well as to decide which of the 17 analytes they wish to target.BIOCRATES AbsoluteIDQ® Steroid Reagents permit simultaneous reliable quanti-fication of as many as 17 steroids, using LC-MS/MS. The reagents are designed for use in combination with „home-brewed“ LC-MS methods.

- AbsoluteIDQ® Steroid Calibrators- AbsoluteIDQ® Steroid Quality Controls- AbsoluteIDQ® Steroid Internal Standards- AbsoluteIDQ® Steroid Testmix

35

Data analysis

Sample measurement service

Biocrates has developed a standardized, quality-controlled, fully integrated technology platform for targeted metabolomics – the system-atic identification and quantification of endogenous metabolites in body fluids and tissues. Our targeted metabolomics approach enables immediate identification of more than 630 endogenous metabolites of different classes, measurement of their absolute concentrations, and mapping to their respective pathways.

Through these Metabolic Phenotyping Services we can help you to identify relevant metabolic biomarkers, accelerate your understanding of biochemical pathways and provide important information of the metabolomic state of a cell, tissue or living organism. Our technology platform offers mass spectrometry-based (FIA-, LC-MS/MS and GC-MS) quantification of metabo-lites through multiple reaction monitoring (MRM) using triple quadrupole state of the art mass spectrometers. For various bio-logical matrices we have established effective metabolite extraction methods and standardized sample preparation protocols.

We offer:

• High-throughput determination of the absolute

concentration of endogenous metabolites

• Broad analytic portfolio of approximately 630

metabolites and 14 metabolite classes

• Small sample volumes due to mass spectrometry

based techniques

• Short turn-around times (4 - 6 weeks from sample

transfer to report)

• Statistical Analysis

• Biochemical interpretation

• Support in study design

• Experience with broad panel of biological matrices

Data analysis

Sample measurement service

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