CLINICAL RESEARCH APPLICATIONS GUIDE

Transcription

1 CLINICAL RESEARCH APPLICATINS GUIDE

2 More throughput Greater sensitivity Reduced costs Less downtime Your job has never demanded more from you. Doctors need accurate results yesterday. You need to minimize costs while increasing the number of samples run. New tests require months and months of design and validation. Phenomenex is committed to providing complete solutions that meet your needs. From sample preparation, to chromatographic separation to detection, we are constantly working to achieve greater sensitivity, shorter run times and improved separations that that your lab can rely on. Since 1982, Phenomenex has grown to become a leading provider of advanced technology solutions for separation science techniques in the areas of High-Performance Liquid Chromatography (HPLC/UHPLC) Gas Chromatography (GC) Sample Preparation 2 For Research Use nly. Not for use in diagnostic procedures. Phenomenex 212 Inc. All rights reserved.

3 LC/MS and LC/MS/MS Applications Aldosterone... 4 Catecholamines... 6 Digitoxin and Digoxin... 7 Estrogens... 8 Gabapentin... 1 Histamine Metanephrine and Normetanephrine Nicotine and its Metabolites Methylmalonic Acid (MMA) Pain Panel H-Progesterone Steroids Panel Testosterone H-Vitamin D 2 and D H-Vitamin D 3 and 3-epi-2-H-Vitamin D Vitamin B6 (Water Soluble) Vitamins (Fat Soluble) Vitamin A... Vitamin D... Vitamin E... Vitamin K... Bath Salts... 3 EtG and EtS GC/MS Application Very Long Chain Fatty Acids Solutions HPLC Columns Solid Phase Extraction (SPE) Products PhenoLogix SM Method Development Method Improvement Validation Services This icon indicates an AB SCIEX imethod Application featuring Phenomenex products is available for this analysis. imethods are the easiest way to get started on an LC/MS/MS method if you are using Cliquid Software. Contact Phenomenex or AB SCIEX for more details. 3

4 Aldosterone verproduction of Aldosterone in the body can lead to primary hyperaldostronism and hypertension. Clinical monitoring of Aldosterone in serum is one of the methods used to understand the cause of hypertension as well as to apply an effective and proper treatment. H H H H H Method Highlights Excellent sensitivity down to 1 ng/dl Aldosterone SAMPLE PREPARATIN Summary: Part No.: Strata -X-A 6 mg/3 ml cartridges were utilized to extract Aldosterone from serum samples. 8B-S123-UBJ Also available in 96-well format SAMPLE PRETREATMENT 1. Into individually labeled test tube combine. ml serum sample (or calibrator or QC sample),1 ml 2 mm ammonium bicarbonate (ph ) and.1 ml working internal standard solution. 2. Proceed to SPE procedure SPE PRCEDURE 1. Condition: 3 ml 1 % Methanol 2. Equilibrate: 3 ml 2 mm Ammonium bicarbonate 3. Load: Pretreated sample 4. Wash 1: 3 ml 2 mm Ammonium bicarbonate. Wash 2: 3 ml 2 % Methanol in Water 6. Dry: min at high vacuum 7. Elute: 2 x 1 ml Ethyl Acetate/Isopropanol/NH 4 H (7:2:1) 8. Evaporate: to - C under a gentle nitrogen stream 9. Reconstitute: 1 µl of Methanol/Water (3:7) containing ~1 ppm Estriol 4

5 Aldosterone LC/MS/MS METHD Analyte Q1 Q3 Aldo Aldo IS (Aldo-D7) Chromatogram of an extracted serum standard. 1.3e4 1.2e4 1.1e4 1e Column: Kinetex 2.6 µm C18 Dimensions: x 3. mm Part No.: B-4496-Y Guard: SecurityGuard ULTRA guard cartridge system Part Nos.: AJ-9 (holder) AJ-877 (C18 cartridges) Mobile phase A: DI Water B: Acetonitrile/Methanol (:) Flow Rate:. ml/min Column Temp: Ambient Injection Volume: 3 µl Gradient : Time (min) Sample: 1. Aldosterone 2. Aldosterone-D7 % B App ID min A typical calibration range from 1-1 ng/dl. Corr Coeff = Tip Transferable to a 2 mm ID column at.4 ml/min Analyte Conc. / IS Conc. Questions or Requests? Contact ClinicalResearch@phenomenex.com for questions on applications or to request additional applications.

6 Catecholamines H Catecholamines are hormones produced by the adrenal glands that are released into the blood during times of stress. Their measurements can aid in the discovery of tumors of the adrenal glands such as pheochromocytoma and the cause hypertension. H H (R) NH 2 Method Highlights Norepinephrine Fast run time and excellent separation with Kinetex core-shell HPLC/ UHPLC columns H H N Extend column lifetime, minimizing down time and costs, with SecurityGuard ULTRA guard cartridge system H H HPLC METHD Column: Kinetex 2.6 µm C18 Dimensions: 1 x 4.6 mm Part No.: F-4462-E Guard: SecurityGuard ULTRA guard cartridge system Part No.: AJ-9 (holder); AJ-8768 (C18 cartridges, 3/pk) Mobile Phase: A: mm Ammonium formate buffer, ph = 3 / Methanol (97:3) Elution Type: Isocratic Flow Rate: 1.2 ml/min Detection: 28 nm Temperature: Ambient Sample: 1. Norepinephrine 2. Epinephrine 3. L-DPA 4. Dopamine. Serotonin H H Epinephrine Dopamine NH mau 2 NH 2 H N H Serotonin App ID min Questions or Requests? Contact ClinicalResearch@phenomenex.com for questions on applications or to request additional applications. 6

7 Digitoxin and Digoxin in Plasma Digitoxin and Digoxin are the active compounds in some drugs used to treat cardiac failure. Both compounds have a narrow therapeutic index, making therapeutic monitoring essential to effective patient treatment. H H H H H H H H H H H Method Highlights Digitoxin Excellent sample clean up with Strata -X SPE Fast run time and excellent separation with Kinetex core-shell HPLC/ UHPLC columns H H H H H H H H H H H H SAMPLE PREPARATIN Summary: Strata-X 3 mg/3 ml cartridges were used to extract the analytes from plasma. Part No.: 8B-S1-TBJ SPE PRCEDURE 1. Condition: 2 ml 2 ml/min 2. Equilibrate: 2 ml 1 mm Ammonium 2 ml/min 3. Load: sample 4. Wash: 1 ml 1 mm Ammonium acetate/1 mm Ammonium acetate 2 ml/min. Dry: 1 minutes at full vacuum 6. Elute: 2 ml 1 ml/min Digoxin Analyte Q1 Q3 Digoxin leandrin (IS) Digitoxin Also available in 96-well format LC/MS/MS ANALYSIS Column: Kinetex 2.6 µm C8 1 Å Dimensions: x 2.1 mm Part No.: B-4497-AN Mobile Phase: A: 1 mm Ammonium acetate B: 1 mm Ammonium acetate in Methanol Gradient: Time (min) % B Flow Rate: 4 μl/min Temperature: 3 C Detection: 3 C Sample: 1. Digoxin 2. leandrin (IS) 3. Digitoxin min App ID

8 Estrogens Estrogens are a group of steroids that are the primary female sex hormones. The three major estrogens in women are estrone (E1), estradiol (E2), and estriol (E3). Method Highlights 6-minute run time Method can be used for quantitation with a linear range from to pg/ml Long column lifetime under high ph conditions with Gemini TWIN technology HPLC columns Estrone Estradiol LC/MS/MS ANALYSIS Method 1: Estrone and Estradiol Column: Gemini 3 µm C6-Phenyl Dimensions: x 2. mm Part No.: B-4443-B Mobile Phase: A: Water +.1 % of. N Ammonium hydroxide B: Methanol +.1 % of. N Ammonium hydroxide Flow Rate:. ml/min Gradient: Time (min) Detector: API % B Analyte Q1 Q3 Estrone Estradiol d4-estrone d3-estradiol Estriol imethod Available Contact AB SCIEX or Phenomenex for details AB SCIEX imethod Test for Estrogens Version 1. for Cliquid Software Method 2: Estriol Column: Gemini 3 µm C6-Phenyl Dimensions: x 2. mm Part No.: B-4443-B Mobile Phase: A: Water +.1 % of. N Ammonium hydroxide B: Methanol +.1 % of. N Ammonium hydroxide Flow Rate:. ml/min Gradient: Time (min) % B Analyte Q1 Q3 Estriol D3-Estriol

9 Estrogens Method 1. Example chromatogram for 1 pg/ml of Estrone and Estradiol generated on an API system in a six-minute run. App ID 263 Method 2. Example chromatogram for 3 pg/ml of Estriol generated on an API system in a six minute run Estriol S/N = 4 26 D 3 -Estriol App ID min min Representative calibration curve for Estrone. The assay was linear across the concentration range of - pg/ml. Representative calibration curve for Estradiol. The assay was linear across the concentration range of - pg/ml Analyte Area / IS Area Analyte Area / IS Area Analyte Conc. / IS Conc. Analyte Conc. / IS Conc. Representative calibration curve for Estriol. The assay was linear across the concentration range of 1- pg/ml Analyte Area / IS Area Questions or Requests? Contact ClinicalResearch@phenomenex.com for questions on applications or to request additional applications. Analyte Conc. / IS Conc. 9

10 Gabapentin Gabapentin is a pharmaceutical drug which has a wide range of uses including the treatment of neuropathic pain, seizures, menopause and treatment of methamphetamine, cocaine, and alcohol addiction. H NH 2 Method Highlights Gabapentin Fast run time with Kinetex core-shell technology HPLC/UHPLC columns Excellent linearity from. µg/ml-2 µg/ml SAMPLE PREPARATIN 1. Transfer: 1 µl of plasma to a. ml centrifuge tube 2. Add: 1 µl of IS/1 %TCA solution to the centrifuge tube, mix briefly 3. 14, rpm for 1 min 4. Transfer: 1 µl of the supernatant to an autosampler vial and QS to 1 ml with water. At this point the sample is ready for analysis. 1

11 Gabapentin LC/MS/MS ANALYSIS Analyte Q1 Q3 Gabapentin Gabapentin D4-Gabapentin Gabapentin extracted from plasma using protein precipitation with trichloroacetic acid at a concentration of 1 µg/ml e 2.e 1.8e 1.6e 1.4e 1.2e 1.e 8.e4 6.e4 Column: Kinetex 2.6 µm C18 1 Å Dimensions: x 2.1 mm Part No.: B-4462-AN Mobile Phase: A: mm Ammonium formate in water B: Acetonitrile Gradient: Time (min) min 1. min 3. min 3.1 min % B Flow Rate:.4 ml/min Injection Volume: 2. µl Temperature: 2 C (column) Detector: API 3 Sample: 1. D4 Gabapentin 2. Gabapentin 4.e4 2.e4 App ID min Gabapentin calibration curve from. µg/ml-2 µg/ml extracted from plasma using protein precipitation with trichloroacetic acid Analyte Area / IS Area Questions or Requests? Contact ClinicalResearch@phenomenex.com for questions on applications or to request additional applications Analyte Conc. / IS Conc. 11

12 Histamine Histamine is involved in immune responses and mediating many important physiological functions such as the regulation of the immune system, nervous system and the secretion of gastric acids. Histamine is formed by the decarboxylation of L-Histidine, an essential amino acid. L-Histidine Method Highlights Good retention and separation using Luna HILIC HPLC columns Excellent sample clean up with Strata -X-CW SPE SAMPLE PREPARATIN Sample prep Histamine was extracted from plasma using Strata-X-CW, 3 mg/3 ml cartridges Part No.: 8B-S3-TBJ Histadine decarboxylase SAMPLE PRETREATMENT 1. Add 1.1 ml IS solution containing 4 ng/ml histmine-d4 and I-methylhistamine-d3 prepared in DI water to µl sample, blank, or plasma 2. Proceed to SPE procedure Histamine SPE PRCEDURE 1. Condition: 1 ml 1 % Methanol 2. Equilibrate: 1 ml DI Water 3. Load: pretreated sample 4. Wash 1: 2 ml DI Water. Wash 2: 2 ml Methanol 6. Dry: 2 min at high vacuum level, ~2-3 in Hg 7. Elute: 2 x 2 µl portions of initial mobile phase composition containing B/A (87:13), fortified to contain % Formic acid 8. Inject: 2 µl on column Also available in 96-well format The sample flow through the SPE bed was kept at a rate of 1.-2 ml/min during all stages of the extraction. 12

13 Histamine LC/MS/MS ANALYSIS Analyte Q1 Q3 Histamine Histamine M-Histamine M-Histamine N-M-Histamine N-M-Histamine Histamine-D Methyl-Hist-D e e4 3.2e4 2.8e4 2.4e4 2.e4 1.6e e min 1.4e e4 1.e min Column: Luna HILIC 3 µm Dimensions: 1 x 2. mm Part No.: D-4449-B Mobile Phase: A: Acetonitrile/DI Water/1 mm Ammonium formate, ph 3.22 (:4:) B: Acetonitrile:1 mm Ammonium formate, ph 3.22 (9:) Flow Rate:.6 ml/min Column Temp: Ambient Injection Volume.: 2 µl Gradient: Time (min) % B Sample: 1. 1-M-Histamine 2. N-M-Histamine 3. Histamine 4.e e4 3.2e4 2.8e4 2.4e4 2.e4 1.6e4 1.2e min App ID 26 Histamine calibration curve, Corr. Coeff.=.9994 Analyte Area / IS Area min Analyte Conc. / IS Conc. Questions or Requests? Contact ClinicalResearch@phenomenex.com for questions on applications or to request additional applications. 13

14 Metanephrine and Normetanephrine Metanephrine and Normetanephrine are metabolites of epinephrine and norepinephrine. Their measurements are key in the identfication of tumors of the adrenal glands such as pheochromocytoma, which can be associated with hypertension. Method Highlights Samples were extracted from plasma to reduce interferences and suppression using Strata -X-CW SPE. Luna HILIC HPLC columns provide good retention for these polar compounds H NH 2 H Metanephrine SAMPLE PREPARATIN Sample Prep: Free Metanephrines (unconjugated) were extracted and concentrated from plasma using 3 mg Strata-X-CW weak cation-exchange polymeric resin in a 96-well plate Part No.: 8E-S3-TGB Normetanephrine SPE PRCEDURE 1. Condition: 4 µl Methanol/Acetonitrile (:) 2. Equilibrate: 4 µl Water 3. Load: µl plasma diluted with 1 ml Water 4. Wash: 8 µl Water followed by 2 ml Methanol/Acetonitrile (:). Dry: 2 minutes at 1 Hg 6. Elute: 2x 2 µl of % Formic acid (freshly prepared) in Acetonitrile/Methanol (:) 7. Dry down: to dryness under stream of nitrogen at 4 C and reconstitute in 1 µl Acetonitrile/1 mm Ammonium formate, ph 3.2 (9:) 8. Cap: reconstituted extract immediately to prevent evaporation Also available in 96-well format 14

15 Metanephrine and Normetanephrine LC/MS/MS METHD HILIC is generally performed using mobile phases containing high concentrations (>7 v/v %) of Acetonitrile, which are ideally suited for use with MS detection. Under HILIC conditions the more polar compounds elute later in the chromatographic run, which is exactly opposite to what is observed under reversed phase conditions. Analyte Q1 Q3 MN MN-d NMN NMN-d e4 7.e4 6.e4.e4 4.e4 3.e4 2.e4 1.e4 Column: Luna 3 µm HILIC Dimensions: x 2. mm Part No.: B-4449-B Mobile Phase: A: Acetonitrile/1 mm Ammonium formate, ph 3.2 (9:) B: Acetonitrile/Water/1 mm Ammonium formate, ph 3.2 (:4:) Flow Rate:.4 ml/min Injection vol: 3 µl of reconstituted extract Gradient: Time (min) min % B 1 1 Sample: 1. Metanephrine (MN) 2. Metanephrine-d3 (MN-d3) 3. Normetanephrine (NMN) 4. Normetanephrine-d3 (NMN-d3) 7.8e4 7.e4 6.e4.e4 4.e4 3.e4 2.e Metanephrine-d / e4 3.e4 3.e4 2.e4 2.e4 1.e4 1.e Normetanephrine-d / e min min Metanephrine 18.4/ min Normetanephrine 166.4/134.2 App ID min Questions or Requests? Contact ClinicalResearch@phenomenex.com for questions on applications or to request additional applications. 1

16 Nicotine and its Metabolites Nicotine is the most abundant alkaloid present in all tobacco products. Determination of nicotine metabolism / pharmacokinetics provides a useful tool for estimating uptake of nicotine and tobacco-related toxicants, for understanding the pharmacologic effects of nicotine and nicotine addiction, and for optimizing nicotine dependency treatment. Tobacco products also contain other alkaloids that can serve as unique markers of tobacco use. Two examples are anabasine and nornicotine, which are present in tobacco products, but not in nicotine replacement therapies. Nicotine Method Highlights Fast run time (<6 minutes) Resolution of alkaloids that can confirm tobacco use Anabasine SAMPLE PREPARATIN Summary: The samples were extracted using Strata -X-C 6 mg/3 ml tubes Part No.: 8B-S29-UBJ 1. Dilute. ml urine with. ml of 2 mm Ammonium acetate, ph Add 1 µl internal standard 3. Proceed to SPE procedure Also available in 96-well format SPE PRCEDURE 1. Condition: 2 ml Methanol (1-2 ml/min) 2. Equilibrate: 2 ml Ammonium acetate buffer 3. Load:. ml diluted urine sample 4. Wash 1: 2 ml Ammonium acetate buffer. Wash 2: 2 ml 3 % Methanol 6. Dry: > 1 Hg for min to remove residual water 7. Elute: 2 x 2. ml 1. % Ammonium hydroxide/methanol solution 8. Dry down: Nitrogen gas at C 9. Reconstitute: µl of Acetonitrile/2 mm Ammonium bicarbonate (1:9) Request our new nicotine tech note! 16

17 Nicotine and its Metabolites LC/MS/MS METHD Nicotine, Cotinine, 3-Hydroxycotinine, Nornicotine and Anabasine analysis (1 ng/ml urine extracted standard) 1.6e 1.e 1.4e 1.3e 1.2e 1.1e 1.e 9.e4 8.e4 7.e4 6.e4.e4 4.e4 3.e4 2.e4 1.e Column: Gemini NX-C18, 3 µm Dimensions: 1 x 2. mm Part No.: D-443-B Mobile Phase: A: 2 mm Ammonium Bicarbonate B: 1 % Acetonitrile Gradient: Time (min) min % B Flow Rate:. ml/min Injection Volume: 1 µl Temperature: 2 C Sample: 1. Nornicotine 2. 3-H-Cotinine 3. Anabasine 4. Cotinine. Nicotine App ID 261 Analyte Q1 Q3 Nicotine Nicotine-d Cotinine Cotinine-d Nornicotine Nornicotine-d H-Cotinine H-Cotinine-d Anabasine Standard curves from 1 ng/ml to ng/ml for Nicotine and Nornicotine Nicotine NH Analyte Area/ IS Area R =.9997 H Analyte Conc./ IS Conc. Questions or Requests? Contact ClinicalResearch@phenomenex.com for questions on applications or to request additional applications. 17

18 Methylmalonic Acid (MMA) Methylmalonic Acid (MMA) levels are tested to monitor Vitamin B12 deficiency, which is often related to the metabolic disorder methylmalonic acidemia. H H Method Highlights 2 minute run time and excellent peak shape using Gemini HPLC columns Methylmalonic Acid SAMPLE PREPARATIN Summary: The samples can be extracted from urine or serum using Strata -X-AW 3 mg/1 ml Part No.: 8B-S38-TAK SPE PRCEDURE 1. Into individually labeled 1. ml conical micro-centrifuge tubes combine. ml 2 mm Ammonium formate, µl IS and 1 µl blank, standard, or sample 2. Activate the SPE cartridge with 1 ml 1 % Methanol 3. Equilibrate the SPE cartridge with 1 ml 2 mm Ammonium formate 4. Load sample and proceed with elution. Wash the SPE cartridge with. ml % Methanol 6. Dry the SPE bed under high vacuum for -1 min 7. Elute the analyte with 2 x.6 ml 2 % NH 4 H in Methanol 8. Evaporate the tubes in a concentrator at 4- ºC 9. Remove the tubes and resuspend the residue in 2 µl.1% Formic acid 1. Inject 1 µl on column LC/MS/MS METHD 2.6e 2.4e 2.2e 2.e 1.8e 1.6e 1.4e 1.2e 1.e 2 Column: Gemini 3 µm C18 Dimensions: 1 x 3. mm Part No.: D-4439-Y Mobile Phase: A:.1 % Formic acid in DI Water B:.1 % Formic acid + 1 mm Ammonium Formate in Methanol Gradient: Time (min) % B Flow Rate:.7 ml/min Injection Volume: 1 µl Temperature: 4 C Sample: 1. Succinic Acid 2. Methyl Malonic Acid (MMA) 8.e4 6.e4 4.e4 2.e4 1 App ID min 18

19 Pain Control and Illicit Drug Panel Pain management drugs are among the most abused drugs today. LC/MS/MS is a key technique for determining which drugs are being abused, whether through illicit or prescribed use. Method Highlights Morphine Excellent separation of isobaric compounds Choose from 2 HPLC/UHPLC stationary phases (C18 and PFP) to optimize separations for your sample SPE PRCEDURE: Summary: Strata -X-Drug B 33 µm Polymeric Strong Cation, 6 mg / 6 ml, Tube, 3/pk Part No.: 8B-S128-UCH Codeine The solvent volumes shown below are for a 6 mg bed mass. The solvent volumes will need to be adjusted for a smaller or larger bed mass. 1. Conditioning: No conditioning required. 2. Load: i. To 2 ml urine sample, add µl of conc. HCl. ii. Heat at 9 C for 2 hours. iii. Add 2 ml of 2 mm Sodium Acetate Buffer (ph 4.). iv. Add 1 ml of 6 N KH. Vortex. v. Centrifuge for mins at rpm. vi. Verify ph of sample is between vii. Load pre-treated sample directly onto SPE cartridge. 3. Wash: i. 2 ml of 1 mm Sodium acetate (ph.) buffer ii. 2mL of Methanol 4. Elute 2 ml of Ethyl acetate:isopropanol:nh 4 H(7:2:1). Evaporate: to dryness under a stream of nitrogen at C. 6. Reconstitute: 1 ml of 1 % methanol. 7. Inject: μl on HPLC / Mass Spectrometer (MS). xycodone H N Methamphetamine For serum/plasma samples: 1. Pre-treatment: Dilute plasma sample 3:1 with 1 % acetic acid. Vortex. Centrifuge for minutes at 3 g. Load directly onto Strata-X-Drug B sorbent, and proceed to step 3 of above SPE procedure. No conditioning required. For saliva samples: 1. Pre-treatment: Dilute saliva sample 3:1 with 1% acetic acid. Vortex. Centrifuge for minutes at 3 g. Load directly onto Strata-X-Drug B sorbent., and proceed to step 3 of above SPE procedure. No conditioning required. Lorezepam N H Benzoylecgonine 19

20 Pain Control and Illicit Drug Panel Kinetex 2.6 µm PFP, x 3. mm.e6.e6.e6 4.e6 4.e6 3.e6 3.e6 Column: Kinetex 2.6 µm PFP Dimensions: x 3. mm Part No.: B-4477-Y Mobile Phase: A: 1 mm Ammonium formate B: Methanol Gradient: Time (min) Flow Rate:. ml/min Injection Volume: 2 µl Temperature: 4 C % B e6 2.e6 1.e6 1.e6.e min App ID 293 Resolution of Critical Isobars 1.e4 1.4e4 1.3e4 1.2e4 1.1e4 1.e Morphine/H-Morphine min Codeine/H-Codeine min ID Q1 Q3 Morphine xymorphone Hydromorphone Codeine xycodone Amphetamine Naltrexone MAM Hydrocodone Methamphetamine MDA Benzoylecgonine Naloxone MDMA ID Q1 Q3 MDEA Norfentanyl Meprobamate Tramadol Normeperidine Meperidine PCP Norbuprenorphine EDDP Fentanyl Flurazepam Norpropoxyphene Clonazepam Flunitrazepam ID Q1 Q3 a-hydroxyalprazolam Propoxyphene Methadone Sufentanil Carisoprodol xazepam Lorazepam Alprazolam Temazepam Nordiazepam Midazolam Buprenorphine Diazepam

21 Pain Control and Illicit Drug Panel Kinetex C µm, x 3. mm 2.e6 1.9e6 1.8e6 1.7e6 1.6e6 1.e6 1.4e6 1.3e6 1.2e6 1.1e6 1.e6 9.e 8.e 7.e 6.e.e 4.e 3.e 2.e 1.e Column: Kinetex 2.6 µm C18 Dimensions: x 3. mm Part No.: B-4462-Y Mobile Phase: A: 1 mm Ammonium Formate B: Methanol with.1 % Formic Acid Gradient: Time (min) Flow Rate:. ml/min Injection Volume: 1 µl Temperature: Ambient min % B 1 1 App ID 2689 ID Q1 Q3 xymorphone Morphine Hydromorphone Amphetamine Methamphetamine xycodone MDA MDMA Codeine Naltrexone MDEA MAM Hydrocodone Benzoylecgonine Norfentanyl Tramadol Naloxone Normeperidine Meperidine Meprobamate PCP Norbuprenorphine EDDP Norpropoxyphene Clonazepam Flunitrazepam a-hydroxyalprazolam Carisoprodol xazepam Methadone Lorazepam Fentanyl Flurazepam Alprazolam Temazepam Propoxyphene Nordiazepam Diazepam Midazolam Sufentanil Buprenorphine Resolution of Critical Isobars Codeine/Hydrocodone 2.4e 2.e 1.e. 3.e 2.e 1.e. 1.e6 9.e 8.e 7.e 6.e.e 4.e 3.e 2.e 1.e min Morphine/Hydromorphone/Norhydrocodone min xymorphone/dihydrocodeine/noroxycodone min 21

22 17-H-Progesterone Measurements of levels of 17-hydroxyprogesterone are useful in the evaluation of suspected congenital adrenal hyperplasia (CAH). CAH leads to excess production of androgen, resulting in male characteristics to appear earlier than normal in men or more prominently than usual in females. Method Highlights 17-H-Progesterone Excellent peak shape and linearity using Gemini NX-C18 HPLC columns SAMPLE PREPARATIN Summary: Strata -X-A 3 mg/3 ml SPE tubes were used to extract the samples from human serum Part No.: 8B-S123-TBJ SAMPLE PRETREATMENT 1. Dilute.2 ml of sample with 1 ml DI Water and.1 ml IS solution (1 ng/ml Testo-D3 and 3 ng/ml 17-H-Prog-D8 in Methanol/DI Water (:)) 2. Proceed to SPE procedure SPE PRCEDURE 1. Condition: 1 ml 1 % Methanol 2. Equilibrate: 1 ml DI Water 3. Load: pretreated sample 4. Wash:.6 ml % Methanol. Dry: under high vacuum for 2-3 min 6. Elute: 2 x.6 ml 1 % Methanol 7. Evaporate: to dryness at - C under gentle nitrogen stream 8. Reconstitute: 2 µl 2 % Acetonitrile in.1 % Formic acid Also available in 96-well format 22

23 17-H-Progesterone LC/MS/MS ANALYSIS Analyte Q1 Q3 17-H-Progesterone (17-H-1) 19.2 (17-HP-2) 17-H-Progesterone-D µg/l standard 2.4e4 2.2e4 2.e4 1.7e4 1.6e4 1.4e4 1.2e4 1.e4 1 2 Column: Gemini NX-C18 3 µm Dimensions: x 2. mm Part No.: B-443-B Mobile Phase: A:.1 % Formic acid in DI Water B:.1 % Formic acid in Acetonitrile Flow Rate:.4 ml/min Column Temp: Ambient Injection Volume: 2 µl Gradient: Time (min) % B Sample: 1. Testosterone H-Progesterone App ID min Calibration Data 17-H-Progesterone Analyte Area / IS Area e4 Analyte Conc. / IS Conc. 23

24 Steroids Panel Corticosteroids are an important class of compounds that have uses as therapeutic agents as well as illicit uses. Steroid hormones are widely used throughout the body and are involved in numerous regulatory pathways including immune response, sexual differentiation, and metabolic function. Testosterone Method Highlights Separate 11 steroids from serum in one run on Kinetex HPLC/UHPLC C18 columns Quick sample preparation using protein precipitation Progesterone SAMPLE PREPARATIN Summary: The analytes were extracted from 2 µl of human serum by protein precipitation, diluted, and injected. Part No.: CE-76 For protein precipitatin method details, go to Analyte Q1 Q3 Q3 DHEAS Cortisol Deoxycortisol Androstenedione Estradiol (water loss) Testosterone H-Progesterone DHEA Progesterone Hydroxyvitamin D Corticosterone H H H H Cortisol H H imethod Available Contact AB SCIEX or Phenomenex for details AB SCIEX imethod Test for Steroid Panel Version 1.1 for Cliquid Software TIP: Consider Impact for high throughput protein precipitation. See 24

25 Steroids Panel 1.8E6 Questions or Requests? Contact for questions on applications or to request additional applications. 1.6E6 1.4E6 1.2E6 1.E6 8.E 6.E Column: Kinetex 2.6 µm C18 Dimensions: 1 x 3. mm Part No.: D-4462-Y Mobile Phase: A: Water B: Methanol Flow Rate:. ml/min Gradient table: Time (min) % B E 2.E min App ID 26 Standard calibration curves (n=4) r = r = Testosterone.1-2 ng/ml H-Progesterone.1-2 ng/ml Analyte Area / IS Area %CV < 7.6% % accuracy > 94.% Analyte Area / IS Area %CV < 9.8% % accuracy > 94.6% Analyte Conc. / IS Conc. Analyte Conc. / IS Conc r = r =.991 Analyte Area / IS Area Progesterone.1-2 ng/ml Analyte Area / IS Area Estradiol.1-2 ng/ml %CV < 8.4% % accuracy > 94.4% %CV < 9.88% % accuracy > 94.2% Analyte Conc. / IS Conc Analyte Conc. / IS Conc. 2

26 Total Testosterone Method Highlights Excellent sample cleanup from plasma using Strata -X-A SPE Fast run time in less than 3 minutes using Gemini NX-C18 HPLC columns SAMPLE PREPARATIN Testosterone Summary: Part No.: Total testosterone was extracted from human plasma using Strata-X-A 3 mg/3 ml SPE cartridges 8B-S123-TBJ SAMPLE PRETREATMENT 1. Dilute.2 ml of sample with 1 ml DI water and.1 ml IS solution (2 ng/ml Testosterone-D3 in Methanol) Also available in 96-well format 2. Proceed to SPE Procedure SPE PRCEDURE 1. Conditions: 1 ml 1 % Methanol 2. Equilibrate: 1 ml Water 3. Load: pretreated sample 4. Wash:.6 ml % Methanol. Dry: SPE Bed under vacuum 6. Elute: 2 x.3 ml 1 % Methanol 7. Evaporate: to dryness at - C under a stream of Nitrogen 8. Add:. ml 2 % Hydroxylamine to form oxime and heat for -6 min at 6-6 C 9. Add:.2 ml % Formic acid in DI Water 1. Inject: 2 µl onto the column Note: The quantitation is based on the testosterone-oxime derivative See Performance Characteristics of a Novel Tandem Mass Spectrometry Assay for Serum Testosterone. Kushnir, Mark M, et al. ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 8418, USA. Clin Chem 2: Jan. 26

27 Total Testosterone LC/MS/MS METHD Analyte Q1 Q3 Testosterone Testosterone-D e 3.4e 3.2e 3.e 2.8e 2.6e 2.4e 2.2e 2.e 1.8e 1.6e 3 Column: Gemini NX-C18 3 µm Dimensions: x 2. mm Part No.: B-4439-B Mobile Phase: A:.1 % Formic acid, 1 mm Ammonium formate in DI Water B:.1 % Formic acid, 1 mm Ammonium formate in Acetonitrile Column Temp: C Injection Volume: 2 µl Flow Rate:.4 ml/min Gradient: Time (min) % B Analytes: 1. Testosterone 2. Testosterone-D3 3. Epitestosterone 1.4e 1.2e 1.e 8.e4 6.e4 4.e4 2.e4 2 App ID min Calibration Curve 1 ng/dl 1, ng/dl Analyte Area / IS Area Questions or Requests? Contact ClinicalResearch@phenomenex.com for questions on applications or to request additional applications e4 Analyte Conc. / IS Conc. 27

28 2-H-Vitamin D 2 and D 3 Vitamin D is recognized as an essential nutrient for intestinal absorption of calcium and phosphate and for promoting bone resorption and formation. Deficiency can lead to bone diseases such as rickets and osteomalacia. Vitamin D levels are measured by the total serum concentrations of the two common forms of 2-H Vitamin D, which are 2-H D 2 and 2-H D 3. H Method Highlights Customer-submitted sample run on a multi-channel system H Vitamin D 2 ergocalciferol 4- µl injection volume SAMPLE PREPARATIN 1. Add: µl of the precipitating reagent containing internal standard to a 1. ml centrifuge tube 2. Pipette: 1 µl of serum into a centrifuge tube 3. Vortex: 2 3 seconds 4. Inspect: each tube to ensure no unmixed sample remains in the bottom of the tube. i. A homogeneous mixture is critical ii.. Centrifuge: 1 minutes at 13, rpm If unmixed sample remains at the bottom of the tube, dislodge by inverting and tapping, then re-vortex 6. Transfer: supernatant into sample vial without disturbing the pellet Vitamin D 3 cholecalciferol TIP: Consider Impact for high throughput protein precipitation. See 28

29 2-H-Vitamin D 2 and D 3 LC/MS/MS ANALYSIS Analyte Q1 Q3 2-H D H D H D3-d Sample Chromatogram run on a multichannel system 1.14e4 1.e e4 1.4e4 1.2e4 1.e H D2 2-H D3 2 1 Column: Kinetex 2.6 µm C18 Dimensions: x 4.6 mm Part No.: B-4462-E Mobile Phase: A:. % Formic acid B: mm Ammonium acetate with.1 % Formic Acid in Methanol % B Gradient: Time (min) Flow Rate: 1 ml/min Temperature 3 ºC Sample: 2-hydroxyvitamin D2 (2-H D2) 2-hydroxyvitamin D3 (2-H D3) 2-hydroxyvitamin D3-d6 (2-H D3-d6) min min 6.e4.e4 4.e4 3.e4 2.e4 1.e4 3 2-H D3-d6 (IS) min App ID Request our technical note on 2-H-VitaminD 2 and D 3! TN-1 Questions or Requests? Contact ClinicalResearch@phenomenex.com for questions on applications or to request additional applications. 29

30 2-H-Vitamin D 3 and 3-epi-2-H-Vitamin D 3 Vitamin D (Ergocalciferol, D 2 and Cholecalciferol, D 3 ) has been under intense investigations and its beneficial health effects have been associated with various treatments from treating bone deficiency to a potent anticancer agent. However, isomerization of monohydroxy Vitamin D produces 3-epi (conversion of a-h to ß-H), a diasteromeric form. There is some ambiguity as to the clinical significance of the 3-epi isomer in general population and is currently under investigation. Method Highlights Good separation of 2-H-Vitamin D 3 and 3-epi-2-H-Vitamin D 3 for accurate quantitation using Kinetex 2.6 µm PFP Fast run time in less than minutes LC/MS/MS ANALYSIS Column: Kinetex 2.6 µm PFP Dimensions: 1 x 2.1 mm Part No.: D-4477-AN Temperature: Ambient Mobile Phase: A:.1 % Formic acid in DI Water B:.1 % Formic acid in Methanol Flow Rate:.4 ml/min Gradient: Time (min) % B Request our technical note on 3-epi-2-H Vitamin D 3! TN-113 MRM TRANSITINS Analyte Q1 Q3 H-Vit D H-Vit D3/Epi-D IS (H-D3-2H3) H-Vit D3 (Sec trans) H-Vit D2 (Sec trans)

31 2-H-Vitamin D 3 and 3-epi-2-H-Vitamin D 3 2.4e6 2.2e6 2.e6 1.8e6 2-H-D 3 1.6e6 1.4e6 1.2e6 1.e6 8.e 6.e 3-Epi-2-H-D 3 2-H-D 2 4.e 2.e min App ID 23 H-Vit D 3 Calibration Curve from 2. 1 ng/ml Analyte Area / IS Area Analyte Conc. / IS Conc. App ID 27 H-Vit D 2 Calibration Curve from 2. 1 ng/ml.34 Analyte Area / IS Area Analyte Conc. / IS Conc. App ID 26 31

32 Vitamin B 6 Vitamin B 6 currently refers to six biologically interconvertible 3-hydroxy-2- methylpyridine compounds: pyridoxal (PL), pyridoxine (PN), pyridoxamine (PM), and their respective -phosphate (PLP, PNP, and PMP). f these compounds, PLP is the primary biologically active form of vitamin B 6. H N H H P Method Highlights Vitamin B 6 Gemini -NX HPLC columns provide baseline separation of PLP, 4-PA and PL Accurately quantitate PLP and 4-PA SAMPLE PREPARATIN 1. Thaw plasma samples and sera/plasma spiked calibrators or pre-manufactured calibration standards and controls at ambient temperature. Protect from light. 2. Pipette 4 μl of the blank (Water), calibration standards, controls and plasma specimens into the appropriate labeled 1. ml Eppendorf microcentrifuge tubes. i. Briefly vortex the calibrators and controls immediately prior to sampling. ii. Mix the plasma samples by gentle inversion immediately prior to sampling. iii. Protect the tubes from light 3. Add 3 μl of 2 mg/ml semicarbazide/glycine solution in rapid succession into all the tubes containing samples; cap the tubes, vortex for 1 seconds. 4. Incubate in the dark at room temperature for 3 minutes.. Uncap the tubes; add μl of 2 % meta-phosphoric acid to the controls and patient samples. 6. Recap the tubes and vortex for 3 seconds at room temperature. 7. Centrifuge for minutes at 14, rpm. Note: The relative centrifugal force (RCF)=16, g. 8. Transfer 3 μl of supernatant to a 2 ml amber vial. 9. Cover the vial with lid and place in the autosampler (RT). 1. Inject 3 µl. 32

33 Vitamin B 6 HPLC ANALYSIS Sample chromatogram of semicarbazide derivatized PLP and PL using Gemini 3 µm NX-C18 HPLC column (1 x 4.6 mm I.D.) mau Column: Gemini -NX 3 µm C18 Dimensions: 1 x 4.6 mm Part No.: D-443-E Guard: Security Guard Cartridge C18, 4 x 3. mm I.D. Part No.: AJ-8368 Mobile Phase: A: 2 mm Sodium phosphate and 1. ml Acetic acid in DI water; ph = ~6.; no ph adjustment. B: (7:3) Acetonitrile:Methanol Gradient: Time (min) Flow Rate: 1. ml/min Injection Volume: 3 µl Temperature: 3 C Fluorescence: Detection: Ex 36 Em 4 Sample: 1. PLP 2. 4-PA 3. PL % B App ID min HUMAN SERUM PLP STANDARD CURVE Example Standard Curve from 6.2 nmol/l to 2 nmol/l for PLP in Human Serum (external standard method) y = 6.442x R 2 =.9999 Peak Area Questions or Requests? Contact ClinicalResearch@phenomenex.com for questions on applications or to request additional applications PLP Concentration in Human Serum (nmol/l) 33

34 Vitamins Fat Soluble Fat soluble vitamins play an important role in healthy bone and teeth development, blood clotting, weight loss, and providing antioxidants. After consumption, fat soluble vitamins are absorbed through the intestinal tract, stored in body tissues and remain in the body longer than their water-soluble counterparts. Vitamin K 1 3 H SAMPLE PREPARATIN 1. The individual vitamin samples were prepared as follows: Vitamin A-acetate (2 mg/ml) Dissolve 2 mg of Vitamin A-acetate in 1 ml of Methanol. Vitamin E Vitamin D 3 (2 mg/ml) Dissolve 2 mg of Vitamin D 3 in 1 ml of Methanol. Vitamin E (8 mg/ml) Dissolve 8 mg of Vitamin E in 1 ml of Methanol. Vitamin E-acetate ( mg/ml) Dissolve mg of Vitamin E-acetate in 1 ml of Methanol. Vitamin K 1 ( mg/ml) Dissolve mg of Vitamin K 1 in 1 ml of Acetonitrile (Vitamin K 1 is not soluble in Methanol). 2. Each vitamin sample was diluted with appropriate solvents to 1 mg/ml. Vitamin A 3. 2 µl of each vitamin was mixed to yield the vitamin mix. HPLC ANALYSIS mau Column: Kinetex 2.6 µm C18 Dimensions: 1 x 4.6 mm Part No.: D-4462-E Guard: SecurityGuard ULTRA System Part No.: AJ-9 (holder) AJ-8768 (cartridges, 3/pk) Gradient Isocratic Mobile Phase: Acetonitrile:Methanol (9:) Flow Rate: 1. ml/min Temperature: 2 C Detection: 28nm Analytes: 1. Vitamin A (trans-retinol) 2. Vitamin A acetate 3. Vitamin E(alpha tocopherol) 4. Vitamin D3 (Cholecalciferol). Vitamin E acetate (alpha tocopherol acetate) 6. Vitamin K1 (Phylloquinone) App ID min 34

35 Bath Salts Bath Salts are among the newest designer drugs that target predominantly young people while attempting to stay ahead of existing anti-drug laws. Bath salts are similar to amphetamines in their chemical composition, effects and dangers. MDPV 4.e6 4.e6 Column: Kinetex 2.6 µm C18 1 Å Dimensions: x 3 mm Part No.: B-4462-Y Mobile Phase: A: 1 mm Ammonium formate B: Methanol with.1 % Formic acid Gradient: Time (min) % B 1 1 Flow Rate:. ml/min Temperature: Ambient Detector Info: API 4 Sample: 1. Methylone 2. Mephedrone 3. MDPV 4. Methamphetamine Mephedrone N H 3.e6 3.e6 2.e6 2.e6 1.e6 Methylone N H 1.e6.e 4 App ID min 3

36 Ethyl glucuronide (EtG) and Ethyl sulfate (EtS) Ethyl glucuronide (EtG) and Ethyl sulfate (EtS) are ethanol metabolites that indicate recent ingestion of alcohol. These metabolites can be detected in urine for up to 8 hours after ingestion, providing a more accurate tool for monitoring alcohol use and abuse compared to standard urine alcohol tests or breathalyzers. EtG Method Highlights Excellent retention of EtG and EtS SAMPLE PREPARATIN Summary: The sample was diluted prior to injection. LC/MS/MS ANALYSIS EtS Analyte Q1 Q3 EtG EtS e4 6.e4 6.e4.e4.e4 4.e4 4.e4 Column: Synergi 2. µm Hydro-RP 1 Å Dimensions: 1 x 3 mm ID Part No.: D-4387-Y Mobile Phase: A: 1 mm Ammonium Acetate B: Methanol:Acetonitrile (:) Flow Rate: μl/min Gradient: Time (min) % B Temperature: 2 C Detector: API 4 Sample: 1. EtG (ethyl glucunoride) 2 2. EtS (ethyl sulfate) 3.e4 3.e4 2.e4 2.e4 1.e4 1.e min App ID 1973 Questions or Requests? Contact ClinicalResearch@phenomenex.com for questions on applications or to request additional applications. 36

37 Very Long Chain Fatty Acids by GC/MS Fatty Acid levels can be indicative of the effects of dietary intake, modifications, and supplements on one s health. f particular interest are levels of cis and trans fatty acid levels due to the link between trans fats and cardiovascular disease. pa App ID min Column: SGE BPX7 Phase: 7 % cyanopropyl polysilphenyl-siloxane Dimensions: 2 m x.32 mm x.2 µm Part No.: CG-16 ven Profile: 8 C for 2 min to 13 C/min for 1 min to C/min for 6 min Carrier Gas: Constant Flow Helium, 2.2 ml/min Injection: Split 48:1;.4 2 C Detection: Flame Ionization (FID) (3 C) Liner: AG-71, 4 mm ID FocusLiner Analyst Note: Analytes are 1 ppm in Hexane Analytes: 1. C6 2. C8 3. C1 4. C11. C12 6. C13 7. C14 8. C14:1 cis 9 9. C1 1. C C16:1 cis C C17:1 cis C18 1. C18:1 trans C18:1 cis C18:1 cis C18:2 trans 9, C18:2 cis 9, C18:3 cis 6, 9, C18:3 cis 9, 12, C18:4 Cis 6, 9, 12, C C21:1 cis 11 Chromatogram and conditions courtesy of Masterfoods UK. 37

38 Columns for LC/MS/MS Separations CRE-SHELL TECHNLGY Ultra High Performance on ANY LC System Get sub-2 µm performance and speeds on any LC system Increase your throughput without loss of critical pair resolution Save money by using less solvent due to faster runtimes Core-Shell Technology 1.2 µm Solid Core.23 µm Porous Shell Kinetex 1.7 µm Core-Shell Particle 1.7 µm Reduced diffusion path maximizes efficiency Increased efficiencies compared to traditional fully porous sub-2 µm columns. Typical operating backpressures > 4 bar 1.9 µm Solid Core.3 µm Porous Shell Kinetex 2.6 µm Core-Shell Particle 2.6 µm Reduced diffusion path maximizes efficiency Ultra-high performance on any HPLC or UHPLC system with Kinetex 2.6 µm columns FULLY PRUS PARTICLE SLUTINS The Standard for ph Method Development Rugged, fully porous particles with excellent column lifetimes ph 1-12 stability allows for low and high ph mobile phases to optimize selectivity for ionizable compounds Full Range Selectivity for Reversed Phase Separation 4 unique phases to optimize even the most challenging separations Available in 2. µm and 4 µm particles for high efficiencies ne of the World s Leading HPLC Columns Numerous phases including C18(2), Phenyl-hexyl, PFP(2), HILIC and others Available in 2. µm and 3 µm particles for high efficiencies 38

39 Kinetex Core-Shell Technology rdering Information 2.6 µm Analytical Columns (mm) SecurityGuard ULTRA Cartridges Phases 3 x 4.6 x x x x 4.6 3/pk XB-C18 B-4496-E C-4496-E D-4496-E F-4496-E AJ-8768 C18 A-4462-E B-4462-E C-4462-E D-4462-E F-4462-E AJ-8768 C8 B-4497-E C-4497-E D-4497-E F-4497-E AJ-877 PFP A-4477-E B-4477-E C-4477-E D-4477-E F-4477-E AJ-8773 HILIC A-4461-E B-4461-E C-4461-E D-4461-E F-4461-E AJ-8772 Phenyl-Hexyl B-449-E D-449-E F-449-E AJ-8774 for 4.6 mm ID NEW µm Columns Available! Call for details µm 2.6 µm MidBore Columns (mm) SecurityGuard ULTRA Cartridges Phases 3 x 3. x 3. 7 x 3. 1 x 3. 1 x 3. 3/pk XB-C18 A-4496-Y B-4496-Y C-4496-Y D-4496-Y F-4496-Y AJ-877 C18 A-4462-Y B-4462-Y C-4462-Y D-4462-Y F-4462-Y AJ-877 C8 A-4497-Y B-4497-Y C-4497-Y D-4497-Y F-4497-Y AJ-8777 PFP A-4477-Y B-4477-Y C-4477-Y D-4477-Y F-4477-Y AJ-878 HILIC A-4461-Y F-4461-Y AJ-8779 Phenyl-Hexyl AJ-8781 for 3. mm ID 2.6 µm Minibore Columns (mm) SecurityGuard ULTRA Cartridges Phases 3 x 2.1 x x x 2.1 3/pk XB-C18 A-4496-AN B-4496-AN D-4496-AN F-4496-AN AJ-8782 C18 A-4462-AN B-4462-AN D-4462-AN F-4462-AN AJ-8782 C8 A-4497-AN B-4497-AN D-4497-AN F-4497-AN AJ-8784 PFP A-4477-AN B-4477-AN D-4477-AN F-4477-AN AJ-8787 HILIC A-4461-AN B-4461-AN D-4461-AN F-4461-AN AJ-8786 Phenyl-Hexyl B-449-AN D-449-AN AJ-8788 for 2.1 mm ID 1.7 µm MidBore Columns (mm) SecurityGuard ULTRA Cartridges Phases 3 x 3. x 3. 1 x 3. 3/pk XB-C18 A-4498-Y B-4498-Y D-4498-Y AJ-877 C18 B-447-Y D-447-Y AJ-877 C8 A-4499-Y B-4499-Y D-4499-Y AJ-8777 PFP D-4476-Y AJ-878 HILIC B-4474-Y AJ-8779 Phenyl-Hexyl AJ-8781 for 3. mm ID 1.7 µm Minibore Columns (mm) SecurityGuard ULTRA Cartridges Phases 3 x 2.1 x x x 2.1 3/pk XB-C18 A-4498-AN B-4498-AN D-4498-AN F-4498-AN AJ-8782 C18 A-447-AN B-447-AN D-447-AN F-447-AN AJ-8782 C8 A-4499-AN B-4499-AN D-4499-AN F-4499-AN AJ-8784 PFP A-4476-AN B-4476-AN D-4476-AN F-4476-AN AJ-8787 HILIC A-4474-AN B-4474-AN D-4474-AN AJ-8786 Phenyl-Hexyl B-4-AN D-4-AN F-4-AN AJ-8788 for 2.1 mm ID SecurityGuard ULTRA cartridges require holder, Part No.: AJ-9 If Kinetex core-shell columns do not provide at least an equivalent separation as compared to a competing column of the same phase, return the column with the comparative data within 4 days for a FULL REFUND. SecurityGuard ULTRA Cartridge System The SecurityGuard ULTRA cartridge system protects ultra-high performance columns, like Kinetex, from damaging contaminants and microparticulates. Extend Kinetex column lifetime Simple to use Pressure rated to 2, psi (1,378 bar) Fits virtually all manufacturers columns SECURITYGUARD ULTRA CARTRIDGE HLDERS Part No. Description Unit Price AJ-9 SecurityGuard ULTRA Cartridge Holder ea UHPLC / HPLC SURE-LK HIGH PRESSURE PEEK MALE NUT FITTINGS Part No. Description Unit Price AQ-83 Sure-Lok High Pressure PEEK 1-Pc Nut 1-32, 1/pk for 1/16 in. Tubing, 12, psi (827 bar) AQ-83 Sure-Lok Fitting Tightening Tool, Aluminum ea AQ-83 AQ-83 39

40 Gemini U.S. Patent No. 7, 63, 367 rdering Information 3 µm Microbore, Minibore and Narrow Bore Columns (mm) SecurityGuard Cartridges (mm) Phases x 1. 2 x 2. 3 x 2. x 2. 1 x 2. 1 x 2. x 3. 1 x 3. 1 x 3. 4 x 2.* /1pk C18 B-4439-A M-4439-B A-4439-B B-4439-B D-4439-B F-4439-B B-4439-Y D-4439-Y F-4439-Y AJ-796 C6-Phenyl B-4443-A A-4443-B B-4443-B D-4443-B F-4443-B B-4443-Y D-4443-Y F-4443-Y AJ-7914 $ 479 $ 48 $ 4 $ 699 $ 789 $ 4 $ 73 $ 82 /1pk NX-C18 M-443-B A-443-B B-443-B D-443-B F-443-B B-443-Y D-443-Y F-443-Y AJ-8367 for ID: mm 3 µm Analytical Columns (mm) SecurityGuard Cartridges (mm) Phases 2 x 4. 3 x 4.6 x x x x x 3.* /1pk C18 M-4439-D A-4439-E B-4439-E D-4439-E F-4439-E G-4439-E AJ-797 C6-Phenyl A-4443-E B-4443-E D-4443-E F-4443-E G-4443-E AJ-791 /1pk NX-C18 B-443-E D-443-E F-443-E G-443-E AJ-8368 for ID: mm µm Minibore and Narrow Bore Columns (mm) SecurityGuard Cartridges (mm) Phases 3 x 2. x 2. 1 x 2. 2 x 2. x 3. 1 x 3. 1 x 3. 2 x 3. 4 x 2.* /1pk C18 A-443-B B-443-B F-443-B G-443-B B-443-Y D-443-Y F-443-Y G-443-Y AJ-796 C6-Phenyl A-4444-B B-4444-B F-4444-B B-4444-Y F-4444-Y G-4444-Y AJ-7914 /1pk NX-C18 A-444-B B-444-B F-444-B B-444-Y D-444-Y F-444-Y G-444-Y AJ-8367 for ID: mm µm Analytical Columns (mm) SecurityGuard Cartridges (mm) Phases 3 x 4.6 x x x x x 3.* /1pk C18 A-443-E B-443-E D-443-E F-443-E G-443-E AJ-797 C6-Phenyl A-4444-E B-4444-E D-4444-E F-4444-E G-4444-E AJ-791 /1pk NX-C18 B-444-E D-444-E F-444-E G-444-E AJ-8368 for ID: mm *SecurityGuard Analytical Cartridges require holder, Part No.: KJ-4282 If Gemini analytical columns do not provide at least an equivalent separation as compared to a competing column of the same particle size, similar phase and dimensions, return the column with comparative data within 4 days for a FULL REFUND. 4

41 ) ) ) Solid Phase Extraction for ptimal Clean Up Strata-X U.S. Patent No. 7,119,14 A reversed phase functionalized polymeric sorbent that gives strong retention of neutral, acidic, or basic compounds under aggressive, high organic wash conditions. Material Characteristics Particle Size (µm) 33 Pore Size (Å) 8 Surface Area (m 2 /g) 8 ph Stability Mechanisms of Retention π-π Bonding Hydrogen Bonding Dipole-Dipole Interactions Hydrophobic Interaction )n )n )n Ṅ. Ṅ. Ṅ. STRATA-X rdering Information Sorbent Format Mass Part Number Unit Price Tube 3 mg 8B-S1-TAK 1 ml (1/box) 3 mg 8B-S1-TBJ 3 ml (/box) 6 mg 8B-S1-UBJ 3 ml (/box) 1 mg 8B-S1-EBJ 3 ml (/box) 1 mg 8B-S1-ECH 6 ml (3/box) 2 mg 8B-S1-FBJ 3 ml (/box) 2 mg 8B-S1-FCH 6 ml (3/box) mg 8B-S1-HBJ 3 ml (/box) mg 8B-S1-HCH 6 ml (3/box) Giga Tube mg 8B-S1-HDG 12 ml (2/box) 1 g 8B-S1-JDG 12 ml (2/box) 1 g 8B-S1-JEG 2 ml (2/box) 2 g 8B-S1-KEG 2 ml (2/box) g 8B-S1-LFF 6 ml (16/box) Teflon Tube 2 mg 8B-S1-FBJ-T 3 ml (/box) 2 mg 8B-S1-FDG-T 12 ml (2/box) 96-Well Plate 1 mg 8E-S1-AGB 2 Plates/Box 3 mg 8E-S1-TGB 2 Plates/Box 6 mg 8E-S1-UGB 2 Plates/Box GENERAL EXTRACTIN PRTCL Method written for a 3 mg/1 ml tube 1. Condition 1 ml Methanol followed by 1 ml DI Water 2. Load: Pretreated sample 3. Wash: 1 ml -6 % Methanol in DI Water. 4. Dry: sorbent for 3 seconds. Elute: 2x µl 2 % Formic acid in Methanol or Acetonitrile If Strata-X SPE products do not perform as well or better than your current SPE product of similar phase, mass and size, return the product with comparative data within 4 days for a FULL REFUND. n-line Extraction Cartridge Description Part Number Unit/Box Price Strata-X on-line extraction M-S33-B-CB ea cartridge, 2 x 2. mm Cartridge holder, 2 mm CH-84 ea Instant Method Development Create your wn SPE Method in under 1 minute! 41

42 ) ) ) Strata-X-C U.S. Patent No. 7,119,14 A strong cation-exchange functionalized polymeric sorbent that allows for complete retention of basic compounds with a pk a less than 1., making 1 % organic wash conditions possible. Material Characteristics Particle Size (µm) 33 Pore Size (Å) 8 Surface Area (m 2 /g) 8 ph Stability 1-14 Ionic Capacity 1 meq/g pk a ~ 3 Mechanisms of Retention Strong Cation-Exchange π-π Bonding Hydrophobic Interaction ) = = S - H + ) = = S - H + ) = = S - H + STRATA-X-C rdering Information Format Sorbent Mass Part Number Unit Price Tube 3 mg 8B-S29-TAK 1 ml (1/box) 3 mg 8B-S29-TBJ 3 ml (/box) 6 mg 8B-S29-UBJ 3 ml (/box) 1 mg 8B-S29-EBJ 3 ml (/box) 1 mg 8B-S29-ECH 6 ml (3/box) 2 mg 8B-S29-FBJ 3 ml (/box) 2 mg 8B-S29-FCH 6 ml (3/box) mg 8B-S29-HBJ 3 ml (/box) mg 8B-S29-HCH 6 ml (3/box) Giga Tube mg 8B-S29-HDG 12 ml (2/box) 1 g 8B-S29-JDG 12 ml (2/box) 1 g 8B-S29-JEG 2 ml (2/box) 2 g 8B-S29-KEG 2 ml (2/box) g 8B-S29-LFF 6 ml (16/box) 96-Well Plate 1 mg 8E-S29-AGB 2 Plates/Box 3 mg 8E-S29-TGB 2 Plates/Box 6 mg 8E-S29-UGB 2 Plates/Box GENERAL EXTRACTIN PRTCL Method written for a 3 mg/1 ml tube 1. Condition: 1 ml Methanol followed by 1 ml DI Water 2. Load: Pretreated sample 3. Wash: 1 ml.1 N Hydrochloric acid in DI Water followed by 1 ml.1 N Hydrochloric acid in Methanol 4. Elute: 2x µl % Ammonium hydroxide in Methanol or Acetonitrile If Strata-X SPE products do not perform as well or better than your current SPE product of similar phase, mass and size, return the product with comparative data within 4 days for a FULL REFUND. n-line Extraction Cartridge Description Part Number Unit/Box Price Strata-X-C on-line extraction M-S48-B-CB ea cartridge, 2 x 2. mm Cartridge holder, 2 mm CH-84 ea Instant Method Development Create your wn SPE Method in under 1 minute! 42

43 ) ) ) Strata-X-CW U.S. Patent No. 7,119,14 A weak cation-exchange functionalized polymeric sorbent that allows for complete retention of basic compounds with a pk a greater than 8, including quaternary amines, making 1 % organic wash conditions possible. Material Characteristics Particle Size (µm) 33 Pore Size (Å) 8 Surface Area (m 2 /g) 8 ph Stability 1-14 Ionic Capacity.76 meq/g pk a ~ 4. 3 Mechanisms of Retention Weak Cation-Exchange π-π Bonding Hydrophobic Interaction ) H ) ) H H STRATA-X-CW rdering Information Sorbent Format Mass Part Number Unit Price Tube 3 mg 8B-S3-TAK 1 ml (1/box) 3 mg 8B-S3-TBJ 3 ml (/box) 6 mg 8B-S3-UBJ 3 ml (/box) 1 mg 8B-S3-ECH 6 ml (3/box) 2 mg 8B-S3-FBJ 3 ml (/box) 2 mg 8B-S3-FCH 6 ml (3/box) mg 8B-S3-HBJ 3 ml (/box) mg 8B-S3-HCH 6 ml (3/box) Giga Tube mg 8B-S3-HDG 12 ml (2/box) 1 g 8B-S3-JDG 12 ml (2/box) 1 g 8B-S3-JEG 2 ml (2/box) 2 g 8B-S3-KEG 2 ml (2/box) g 8B-S3-LFF 6 ml (16/box) 96-Well Plate 1 mg 8E-S3-AGB 2 Plates/Box 3 mg 8E-S3-TGB 2 Plates/Box 6 mg 8E-S3-UGB 2 Plates/Box GENERAL EXTRACTIN PRTCL Method written for a 3 mg/1 ml tube 1. Condition: 1 ml Methanol followed by 1 ml DI Water 2. Load: Pretreated sample 3. Wash: 1 ml DI Water followed by 1 ml Methanol 4. Elute: 2x µl % Formic acid in Methanol or Acetonitrile If Strata-X SPE products do not perform as well or better than your current SPE product of similar phase, mass and size, return the product with comparative data within 4 days for a FULL REFUND. n-line Extraction Cartridge Format Part Number Unit/Box Price Strata-X-CW on-line extraction M-S36-B-CB ea cartridge, 2 x 2. mm Cartridge holder, 2 mm CH-84 ea Instant Method Development Create your wn SPE Method in under 1 minute! 43

44 ) ) ) Strata-X-A U.S. Patent No. 7,119,14 A strong anion-exchange functionalized polymeric sorbent that allows for complete retention of weakly acidic compounds with pk a greater than 2, making 1 % organic wash conditions possible. Material Characteristics Particle Size (µm) 33 Pore Size (Å) 8 Surface Area (m 2 /g) 8 ph Stability 1-14 Ionic Capacity.3 meq/g pk a ~ 14 3 Mechanisms of Retention Strong Anion-Exchange π-π Bonding Hydrophobic Interaction ) ) ) CH 3 CH 3 CH 3 N + N + N + CH 3 CH 3 CH 3 STRATA-X-A rdering Information Sorbent Format Mass Part Number Unit Price Tube 3 mg 8B-S123-TAK 1 ml (1/box) 3 mg 8B-S123-TBJ 3 ml (/box) 6 mg 8B-S123-UBJ 3 ml (/box) 1 mg 8B-S123-EBJ 3 ml (/box) 1 mg 8B-S123-ECH 6 ml (3/box) 2 mg 8B-S123-FBJ 3 ml (/box) 2 mg 8B-S123-FCH 6 ml (3/box) mg 8B-S123-HBJ 3 ml (/box) mg 8B-S123-HCH 6 ml (3/box) Giga Tube mg 8B-S123-HDG 12 ml (2/box) 1 g 8B-S123-JDG 12 ml (2/box) 1 g 8B-S123-JEG 2 ml (2/box) 2 g 8B-S123-KEG 2 ml (2/box) g 8B-S123-LFF 6 ml (16/box) 96-Well Plate 1 mg 8E-S123-AGB 2 Plates/Box 3 mg 8E-S123-TGB 2 Plates/Box 6 mg 8E-S123-UGB 2 Plates/Box GENERAL EXTRACTIN PRTCL Method written for a 3 mg/1 ml tube 1. Condition: 1 ml Methanol followed by 1 ml DI Water 2. Load: Pretreated sample 3. Wash: 1 ml 2 mm Ammonium acetate (ph 6-7) followed by 1 ml Methanol 4. Elute: 2x µl % Formic acid in Methanol or Acetonitrile If Strata-X SPE products do not perform as well or better than your current SPE product of similar phase, mass and size, return the product with comparative data within 4 days for a FULL REFUND. Instant Method Development Create your wn SPE Method in under 1 minute! 44

45 ) ) ) Strata-X-AW U.S. Patent No. 7,119,14 A weak anion-exchange functionalized polymeric sorbent that allows for complete retention of acidic compounds with pk a less than, making 1 % organic wash conditions possible. Material Characteristics Particle Size (µm) 33 Pore Size (Å) 8 Surface Area (m 2 /g) 8 ph Stability 1-14 Ionic Capacity.6 meq/g pk a ~ 9 3 Mechanisms of Retention Weak Anion-Exchange π-π Bonding Hydrophobic Interaction ) ) ) NH NH 2 NH NH 2 NH NH 2 STRATA-X-AW rdering Information Sorbent Format Mass Part Number Unit Price Tube 3 mg 8B-S38-TAK 1 ml (1/box) 3 mg 8B-S38-TBJ 3 ml (/box) 6 mg 8B-S38-UBJ 3 ml (/box) 1 mg 8B-S38-EBJ 3 ml (/box) 1 mg 8B-S38-ECH 6 ml (3/box) 2 mg 8B-S38-FBJ 3 ml (/box) 2 mg 8B-S38-FCH 6 ml (3/box) mg 8B-S38-HBJ 3 ml (/box) mg 8B-S38-HCH 6 ml (3/box) Giga Tube mg 8B-S38-HDG 12 ml (2/box) 1 g 8B-S38-JDG 12 ml (2/box) 1 g 8B-S38-JEG 2 ml (2/box) 2 g 8B-S38-KEG 2 ml (2/box) g 8B-S38-LFF 6 ml (16/box) 96-Well Plate 1 mg 8E-S38-AGB 2 Plates/Box 3 mg 8E-S38-TGB 2 Plates/Box 6 mg 8E-S38-UGB 2 Plates/Box GENERAL EXTRACTIN PRTCL Method written for a 3 mg/1 ml tube 1. Condition: 1 ml Methanol followed by 1 ml DI Water 2. Load: Pretreated sample 3. Wash: 1 ml 2 mm Ammonium acetate (ph 6-7) followed by 1 ml Methanol 4. Elute: 2x µl % Ammonium hydroxide in Methanol or Acetonitrile If Strata-X SPE products do not perform as well or better than your current SPE product of similar phase, mass and size, return the product with comparative data within 4 days for a FULL REFUND. Instant Method Development Create your wn SPE Method in under 1 minute! 4

46 Strata-X-Drug B U.S. Patent No. 7,119,14 Strata-X-Drug B is a polymeric strong catiion-exchange SPE sorbent that is designed and quality controlled with Substance Abuse and Mental Health Services Administration (SAMHSA) regulations in mind. 3 EASY T FLLW METHDS Methods are written for 6 mg/6 ml Strata-X-Drug B; however they can be scaled to accommodate smaller or larger sample sizes and sorbent masses. piates, 6-MAM, PCP, and Amphetamines* 1. Condition: No conditioning required 2. Load: Pre-treated urine sample 3. Wash 1: 2 ml of 1 mm Sodium acetate buffer (ph.) 4. Wash 2: 2 ml Methanol. Dry: 1 minutes under full vaccum 6. Elute: 2 ml of Ethyl acetate/isopropanol/ammonium hydroxide (7:2:1) Cocaine Metabolites 1. Condition: No conditioning required 2. Load: Pre-treated urine sample 3. Wash 1: 2 ml of.1 N Hydrochloric acid 4. Wash 2: 2 ml Methanol. Dry: 1 minutes under full vacuum 6. Elute: 2 ml of Ethyl acetate/isopropanol/ammonium hydroxide (7:2:1) * piates, 6-MAM, PCP, and Amphetamines can be extracted simultaneously or separately using the same SPE methodology. Marijuana Metabolites 1. Condition: No conditioning required 2. Load: Pre-treated urine sample 3. Wash 1: 2 ml of 1 mm Sodium acetate buffer (ph.) 4. Wash 2: 2 ml of Acetonitrile/1 mm Sodium acetate buffer (ph.) (3:7). Dry: 1 minutes under full vacuum 6. Elute: 2 ml of Ethyl acetate/isopropanol (8:1) STRATA-X-DRUG B rdering Information Format Sorbent Mass Part Number Unit Price Tube 1 mg 8B-S128-AAK 1 ml (1/box) 1 mg 8L-S128-AAK 1 ml (1/box) 3 mg 8B-S128-TAK 1 ml (1/box) 3 mg 8L-S128-TAK 1 ml (1/box) 3 mg 8B-S128-TBJ 3 ml (/box) 6 mg 8B-S128-UBJ 3 ml (/box) 6 mg 8B-S128-UCH 6 ml (3/box) 6 mg 8B-S128-UCL 6 ml (2/box) Giga Tube 1 mg 8B-S128-EDG 12 ml (2/box) 96-Well Plate Tab-less tube 1 mg 8E-S128-AGB 2 Plates/box 3 mg 8E-S128-TGB 2 Plates/box 6 mg 8E-S128-UGB 2 Plates/box If Strata-X SPE products do not perform as well or better than your current SPE product of similar phase, mass and size, return the product with comparative data within 4 days for a FULL REFUND. Instant Method Development Create your wn SPE Method in under 1 minute! 46

47 An Analytical Support Laboratory PhenoLogix customizes solutions to fit your exact needs and requirements so methods can be transferred efficiently and effectively back into your lab upon completion. Please contact us to get started on your method. or Visit: HW CAN WE BE F SERVICE? PhenoLogix worldwide technical teams are here to provide full service method support and training for you and your lab. ur services include: Method Improvement and ptimization New Method Development Validation and Pre-validation Services n-site Training and Consulting PARTIAL LIST F RESURCES USED IN THE PHENLGIX LAB Agilent, Shimadzu, ACQUITY and JASC HPLC and UHPLC systems API 3 LC/MS/MS API 4 LC/MS/MS API LC/MS/MS 4 Q-TRAP LC/MS/MS Waters Micromass Quattro MS Agilent and Shimadzu GC/MS systems Gilson and PerkinElmer liquid handlers 47