Vitamin Testing APPLICATIONS GUIDE

Vitamin Testing APPLICATIONS GUIDE

Vitamins play an essential role in human health and wellness. While essential, many vitamins cannot be synthesized within the body and must be obtained externally, most commonly from food. Due to poverty, lack of awareness, a person s health, and other factors, many people do not have healthy levels of vitamins in their body and suffer physical and cognitive consequences as a result. Phenomenex strives to continually improve the field of vitamin research testing by offering solutions that improve medical care and increase lab efficiency. Our goal is provide solutions that are: Accurate High-Throughput Reproducible Low Cost 2 For Research Use Only. Not for use in diagnostic procedures. Phenomenex 213 Inc. All rights reserved.

Improve Your Vitamin Analysis Applications Vitamin B 1 from Whole Blood: Thiamine Diphosphate (TDP)... 4-5 Vitamin B 1 from Plasma: Thiamine Monophosphate (TMP) and Thiamine... 6-7 Vitamin B 3... 8-9 Vitamin B 6... 1-11 25-OH-Vitamin D 2 and D 3... 12-13 25-OH-Vitamin D 3 and 3-epi-25-OH-Vitamin D 3... 14-15 HPLC Columns Kinetex Core-Shell HPLC/UHPLC Columns...16 Gemini High ph HPLC Columns...17 Sample Preparation Solutions Impact Protein Precipitation Plates...18 Phree Phospholipid Removal Solutions...19 Strata -X Polymeric Solid Phase Extraction (SPE)...2 Technical Support PhenoLogix SM : Your Analytical Support Laboratory...21 3

Vitamin B 1 from Whole Blood: Thiamine Diphosphate (TDP) Thiamine Diphosphate (TDP) concentration is a useful index for determining thiamine deficiency. Samples are commonly obtained from whole blood because approximately 9 % of total thiamine in blood is in the red blood cells. Deficiency can lead to diseases such as Beriberi and optic neuropathy. Vitamin B 1 Method Highlights Total run time under 4 min Analytical measurement range: 4 nmol/l Good linearity: R 2 =.9991 N NH 2 N + N S O OH P O OH O P OH O Sample Preparation 1. Collect: venous blood into EDTA or heparin containing tubes. 2. Freeze: sample immediately at -7 C or -2 C for up to 7 days. 3. Add: 5 µl of 1 % TCA to 5 µl thawed hemolysates, water blank, calibrators, and controls to precipitate proteins. 4. Vortex: 15 seconds and leave standing on rack for 15 minutes at room temperature. 5. Centrifuge: 14, rpm for 6 minutes at room temperature. Note: The relative centrifugal force (RCF) = 16, g. 6. Transfer: 5 µl supernatant into a glass tube. i. Wash twice with methyl-tert-butyl ether (75 µl) to remove TCA. ii. Vortex for 15 seconds. iii. Centrifuge the tubes at 3,5 rpm for 3 minutes. iv. Remove the tubes and discard the top organic layer. v. Repeat once. 7. Transfer: 5 µl of extract to an autosampler injection vial. 8. Add: 5 µl of water. 9. Add: 5 µl of derivatizing reagent. 1. Vortex: 15 seconds and place the vial on the autosampler at RT. Thiamine Diphosphate 11. Inject: 2 µl of prepared sample. *Derivatizing reagent: Potassium ferricyanide [K 3 Fe(CN) 6 ] was prepared as the following: Dissolve 6 mg of K 3 Fe(CN) 6 in 15 ml 15 % sodium hydroxide solution. Prepare fresh daily. 4

Vitamin B 1 from Whole Blood: Thiamine Diphosphate (TDP) (cont d) LC/MS/MS Method Multi-standards of TDP (-4 nmol/l) injected after sample cleanup mau 7 67.5 65 6 57.5 55 52.5 Column: Gemini 5 µm C18 Dimensions: 5 x 4.6 mm Part No.: B-4435-B Mobile Phase: A: 25 mm Disodium hydrogen phosphate, 1 % Methanol, ph 7. B: 25 mm Disodium hydrogen phosphate, 7 % Methanol, ph 7. Gradient: Time (min) 1 1.5 2.5 2.6 4. % B 12.5 5 9 Flow Rate: 1. ml/min Detection: Fluorescence. Excitation: 375 nm; Emission: 435 nm Sample: TDP (thiamine diphosphate) App ID 2877.5 1 1.5 3.5 3.5 min Standard curve of TDP (Whole blood), 4 nmol/l 9 8 Peak area 7 6 5 4 3 2 1 y =.1969x +.6829 R 2 =.9991 5 1 15 2 25 3 35 4 45 TDP Concentration (nmol/l) App ID 288 Statistic analysis of ten consecutive injections of whole blood sample spiked with TDP and injected after sample cleanup Data summary Whole blood spiked with TDP and injected after sample cleanup Seq. No. RT Area Peak Height Mean 2.634 66.8 12. STDV.52.3521.823 CV (%).2.53.68 Questions or Requests? Contact ClinicalResearch@phenomenex.com for questions on applications or to request additional applications. 5

Vitamin B 1 from Plasma: Thiamine Monophosphate (TMP) and Thiamine Total Thiamine reflects the measurement of Thiamine (Vitamin B 1 ) and Thiamine Monophosphate (TMP). Method Highlights Vitamin B 1 Excellent linearity. TMP R 2 =.9996. Thiamine R 2 =.9986 Sample Preparation 1. Add: 4 µl of methanol to a well of an Impact Protein Precipitation Plate. 2. Add: 1 µl plasma to the well. Mix 3 times with pipette tip (or vortex the whole plate briefly). 3. Wait: 5 minutes. 4. Filter: Under vacuum (5 Hg) for 5 minutes. Ensure that a collection plate is positioned underneath the Impact Protein Precipitation Plate. 5. Transfer: 5 µl to an autosampler vial (or leave sample in collection plate). 6. Add: 1 µl water and 25 µl of derivatizing reagent*. 7. Cover: vial with lid (or collection plate with sealing mat). 8. Vortex: 15 seconds. 9. Put: vial or collection plate into an autosampler. OH O N P OH S O N N NH 2 Thiamine Monophosphate All conditions are at room temperature (RT). *Derivatizing reagent: Potassium ferricyanide [K 3 Fe(CN) 6 ] was prepared as the following: Dissolve 6 mg of K 3 Fe(CN) 6 in 15 ml 15 % sodium hydroxide solution. Prepare fresh daily. LC/MS/MS Method Column: Kinetex 5 µm C18 Dimensions: 1 x 4.6 mm Part No.: D-461-E Mobile Phase: A: 25 mm Disodium hydrogen phosphate, 1 % Methanol, ph 7. B: 25 mm Disodium hydrogen phosphate, 7 % Methanol, ph 7. Gradient: Time (min).25.75 3. 4. 4.5 5. % B 3 25 25 35 6 3 3 Flow Rate: 1 ml/min Temperature: 25 C Backpressure: 15 bar Detection: Fluorescence Detector Excitation: 375 nm; Emission: 435 nm Sample: 1. TMP (Thiamine monophospate) 2. Thiamine 2 nmol/l filtered by an Impact Protein Precipitation Plate mau 1 2 6 58 56 App ID 2875 6 54 2 4 min

Vitamin B 1 from Plasma: Thiamine Monophosphate (TMP) and Thiamine (cont d) 5 nmol/l filtered by an Impact Protein Precipitation Plate mau 56 55 54 1 2 App ID 2881 1 2 3 4 min Standard Curve of TMP Filtered by an Impact Protein Precipitation Plate 35 3 Peak area 25 2 15 y =.1434x -.36 R 2 =.9996 1 5 5 1 15 2 25 TMP Concentration (nmol/l) App ID 2876 Data summary Plasma spiked with TMP Plasma spiked with Thiamine Seq. No. RT Area RT Area Mean 2.62 1.125 4.275 1.65 STDV.148.77.33.756 CV (%).57.7.8 4.58 Questions or Requests? Contact ClinicalResearch@phenomenex.com for questions on applications or to request additional applications. 7

Vitamin B 3 Vitamin B 3, also known as Niacin or Nicotinic Acid is an important part of the human diet. A deficiency of Vitamin B 3 leads to pellagra, which can affect the skin and cause cognitive problems, amongst other symptoms. Method Highlights Fast sample prep using Impact Protein Precipitation Plates Fast run time of under 4 minutes N O Vitamin B 3 OH SAMPLE PREPARATION 1. Place: Impact plate onto a suitable 96-well sample manifold or rotor. 2. Dispense: 3 µl acetonitrile into the wells of the Impact plate. 3. Add: 1 µl of plasma/serum samples to each well of the Impact plate. 4. Mix: 3 times with a pipette tip. 5. Vacuum: to filter the sample and collect the purified filtrate. Ensure that a collection plate is positioned underneath the Impact plate. LC/MS/MS METHOD LC/MS/MS Chromatogram of Nicotinamide and Nicotinic Acid at 1 ng/ml 3.6e5 1 3.4e5 Intensity, cps 3.2e5 3e5 2.8e5 2.6e5 2.4e5 2.2e5 2e5 1.8e5 1.6e5 1.4e5 Column: Gemini 3 µm C18 Dimensions: 1 x 4.6 mm Part No.: D-4439-E Mobile Phase: A:.1 % Formic acid in water B: Methanol Gradient: Time (min) 2.5 2.6 4. % B 1 9 1 1 Flow Rate:.6 ml/min Detection: Electrospray Mass Spec (ESMS) Detector: AB SCIEX API 4 Sample: 1. Nicotinamide 2. Nicotinic Acid 2 1.2e5 1e5 8e4 6e4 4.e4 2.e4.5 1 1.5 2 2.5 3 3.5 min App ID 299 8

Vitamin B 3 (cont d) LC/MS/MS Chromatogram of Nicotinamide and Nicotinic Acid at 1 ng/ml 1 8 Intensity, cps 75 7 65 6 55 5 45 4 Column: Gemini 3 µm C18 Dimensions: 1 x 4.6 mm Part No.: D-4439-E Mobile Phase: A:.1 % Formic acid in water B: Methanol Gradient: Time (min) 2.5 2.6 4. % B 1 9 1 1 Flow Rate:.6 ml/min Detection: Electrospray Mass Spec (ESMS) Detector: AB SCIEX API 4 Sample: 1. Nicotinamide 2. Nicotinic Acid 2 35 3 25 2 15 App ID 296 1 5.5 1. 1.5 2. 2.5 3. 3.5 min Nicotinic Acid Standard Curve, 2-2 ng/ml 1.5e6 1.4e6 1.3e6 1.2e6 1.1e6 1.e6 R 2 =.9996 Area, counts 9.e5 8.e5 7.e5 6.e5 MRM Transitions Used for Data Analysis Peak Name MRM Channel Nicotinamide 123.5 8.1 Nicotinic acid 123.981 8.1 5.e5 4.e5 3.e5 2.e5 1.e5. App ID 291 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 min Concentration, ng/ml 9

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 5 -phosphate (PLP, PNP, and PMP). Of these compounds, PLP is the primary biologically active form of vitamin B 6. HO O N HO OH O P O Method Highlights Vitamin B 6 Gemini NX-C18 HPLC columns provide baseline separation of PLP, 4-PA and PL Accurately quantitate PLP and 4-PA Sample Preparation 1. Thaw patient 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 1.5 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 25 mg/ml semicarbazide/glycine solution in rapid succession into all the tubes containing samples; cap the tubes, vortex for 15 seconds. 4. Incubate in the dark at room temperature for 3 minutes. 5. Uncap the tubes; add 5 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 5 minutes at 14, rpm. Note: The relative centrifugal force (RCF)=16, g. 8. Transfer 35 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. 1

Vitamin B 6 (cont d) HPLC Analysis Sample chromatogram of semicarbazide derivatized PLP and PL mau 75 725 7 675 65 625 1 2 3 Column: Gemini NX-C18 3 µm Dimensions: 1 x 4.6 mm Part No.: D-4453-E Guard: SecurityGuard Cartridge C18, 4 x 3. mm ID 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: Acetonitrile/Methanol (7:3) Gradient: Time (min) 5. 5.1 6. 6.1 7. % B 5 6 95 95 5 5 Flow Rate: 1 ml/min Injection Volume: 3 µl Temperature: 35 C Detection: Fluorescence: Ex 36; Em 45 Sample: 1. PLP (Pyridoxal 5 -Phosphate) 2. PA (4-pyridoxic acid) 3. PL (Pyridoxal) 6 App ID 2684 575 55 1 2 3 4 5 6 min Human Serum PLP Standard Curve Example Standard Curve from 6.25 nmol/l to 2 nmol/l for PLP in Human Serum (external standard method) 14 12 1 y = 65.442x - 154.18 R 2 =.9999 Peak Area 8 6 Questions or Requests? Contact ClinicalResearch@phenomenex.com for questions on applications or to request additional applications. 4 2 5 1 15 2 25 PLP Concentration in Human Serum (nmol/l) App ID 2922 11

25-OH-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 25-OH-Vitamin D, which are 25-OH D 2 and 25-OH D 3. H Method Highlights Customer-submitted patient sample run on a multi-channel system HO Vitamin D 2 ergocalciferol 4-5 µl injection volume Sample Preparation 1. Add: 5 µl of precipitating reagent containing internal standard to a 1.5 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. 5. Centrifuge: 15 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 www.phenomenex.com/impact 12

25-OH-Vitamin D 2 and D 3 (cont d) LC/MS/MS Analysis Analyte Q1 Q3 25-OH D2 395.3 29.3 25-OH D3 383.2 257.2 25-OH D3-d6 389.3 263.3 Patient sample run on a multi-channel system Intensity, cps Intensity, cps 1.14e4 1.e4 8 6 4 2 1.6e4 1.4e4 1.2e4 1.e4 8 6 4 2 25-OH D2 25-OH D3 2 1 Column: Kinetex 2.6 µm C18 Dimensions: 5 x 4.6 mm Part No.: B-4462-E Mobile Phase: A:.5 % Formic acid in Water B: 5 mm Ammonium acetate with.1 % Formic acid in Methanol % B 8 8 1 1 8 Gradient: Time (min).12 3.4 4.8 6 Flow Rate: 1 ml/min Temperature 35 ºC Detection: Mass Spectrometer (MS) Detector: AB SCIEX API 4 Sample: 1. 25-Hydroxyvitamin D2 (25-OH D2) 2. 25-Hydroxyvitamin D3 (25-OH D3) 3. 25-Hydroxyvitamin D3-d6 (25-OH D3-d6) 3.6 3.7 3.8 3.9 4. 4.1 4.2 4.3 4.4 4.5 4.6 min 3.6 3.7 3.8 3.9 4. 4.1 4.2 4.3 4.4 4.5 4.6 min 6.e4 5.e4 25-OH D3-d6 (IS) 3 Intensity, cps 4.e4 3.e4 2.e4 1.e4 3.6 3.7 3.8 3.9 4. 4.1 4.2 4.3 4.4 4.5 4.6 min App ID 19314 View our technical note on 25-OH-Vitamin D 2 and D 3 at www.phenomenex.com/clinical Questions or Requests? Contact ClinicalResearch@phenomenex.com for questions on applications or to request additional applications. 13

25-OH-Vitamin D 3 and 3-epi-25-OH-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-oh to ß-OH), 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 25-OH-Vitamin D 3 and 3-epi-25-OH-Vitamin D 3 for accurate quantitation using Kinetex 2.6 µm PFP HPLC column Fast run time in less than 5 minutes LC/MS/MS Analysis Column: Kinetex 2.6 µm PFP Dimensions: 1 x 2.1 mm Part No.: D-4477-AN Mobile Phase: A:.1 % Formic acid in DI Water B:.1 % Formic acid in Methanol Gradient: Time (min) % B 75 2. 8 3.8 8 3.81 75 6. 75 Flow Rate:.4 ml/min Temperature: Ambient Detection: Mass Spectrometer (MS/MS) Detector: AB SCIEX API 5 View our technical note on 3-epi-25-OH Vitamin D 3 at www.phenomenex.com/clinical MRM Transitions Analyte Q1 Q3 OH-Vit D2 395.3 29.3 OH-Vit D3/Epi-D3 383.2 257.2 IS (OH-D3-2H3) 386.2 257.2 OH-Vit D3 (Sec trans) 383.2 229.1 OH-Vit D2 (Sec trans) 395.3 269.2 14

25-OH-Vitamin D 3 and 3-epi-25-OH-Vitamin D 3 (cont d) 2.4e6 2.2e6 2.e6 1.8e6 25-OH-D 3 1.6e6 Intensity, cps 1.4e6 1.2e6 1.e6 8.e5 6.e5 3-Epi-25-OH-D 3 25-OH-D 2 4.e5 2.e5.5 1 1.5 2 2.5 3 3.5 4 4.5 5 min App ID 23 OH-Vitamin D 3 Calibration Curve from 2.5 1 ng/ml 1.2 1.1 1..9 Analyte Area / IS Area.8.7.6.5.4.3.2.1. 5 1 15 2 25 3 35 4 45 5 55 6 5 7 75 8 85 9 95 1 Analyte Conc. / IS Conc. App ID 27 OH-Vitamin D 2 Calibration Curve from 2.5 1 ng/ml.34 Analyte Area / IS Area.32.3.28.26.24.22.2.18.16.14.12.1.8.6.4.2. 5 1 15 2 25 3 35 4 45 5 55 6 5 7 75 8 85 9 95 1 Analyte Conc. / IS Conc. App ID 26 15

Kinetex Ordering Information 2.6 µm Analytical Columns (mm) SecurityGuard ULTRA Cartridges Phases 3 x 4.6 5 x 4.6 75 x 4.6 1 x 4.6 15 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-4495-E D-4495-E F-4495-E AJ-8774 for 4.6 mm ID NEW 5 µm Columns Available! Call for details 5 µm 2.6 µm MidBore Columns (mm) SecurityGuard ULTRA Cartridges Phases 3 x 3. 5 x 3. 75 x 3. 1 x 3. 15 x 3. 3/pk XB-C18 A-4496-Y B-4496-Y C-4496-Y D-4496-Y F-4496-Y AJ-8775 C18 A-4462-Y B-4462-Y C-4462-Y D-4462-Y F-4462-Y AJ-8775 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 D-4495-Y F-4495-Y AJ-8781 for 3. mm ID 2.6 µm Minibore Columns (mm) SecurityGuard ULTRA Cartridges Phases 3 x 2.1 5 x 2.1 1 x 2.1 15 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 A-4495-AN B-4495-AN D-4495-AN F-4495-AN AJ-8788 for 2.1 mm ID 1.7 µm MidBore Columns (mm) SecurityGuard ULTRA Cartridges Phases 3 x 3. 5 x 3. 1 x 3. 3/pk XB-C18 A-4498-Y B-4498-Y D-4498-Y AJ-8775 C18 B-4475-Y D-4475-Y AJ-8775 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 5 x 2.1 1 x 2.1 15 x 2.1 3/pk XB-C18 A-4498-AN B-4498-AN D-4498-AN F-4498-AN AJ-8782 C18 A-4475-AN B-4475-AN D-4475-AN F-4475-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-45-AN D-45-AN F-45-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 45 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 UHPLC column lifetime Simple to use Pressure rated to 2, psi (1,378 bar) Fits virtually all manufacturers columns SecurityGuard ULTRA Cartridge Holders Part No. Description Unit AJ-9 SecurityGuard ULTRA Cartridge Holder ea UHPLC / HPLC Sure-Lok High Pressure PEEK Male Nut Fittings Part No. Description Unit AQ-853 Sure-Lok High Pressure PEEK 1-Pc Nut 1-32, 1/pk for 1/16 in. Tubing, 12, psi (827 bar) AQ-853 Sure-Lok Fitting Tightening Tool, Aluminum ea AQ-853 AQ-853 16

Gemini U.S. Patent No. 7,563,367 Ordering Information 3 µm Microbore, Minibore and Narrow Bore Columns (mm) SecurityGuard Cartridges (mm) Phases 5 x 1. 2 x 2. 3 x 2. 5 x 2. 1 x 2. 15 x 2. 5 x 3. 1 x 3. 15 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-7596 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 /1pk NX-C18 M-4453-B A-4453-B B-4453-B D-4453-B F-4453-B B-4453-Y D-4453-Y F-4453-Y AJ-8367 for ID: 2.-3. mm 3 µm Analytical Columns (mm) SecurityGuard Cartridges (mm) Phases 2 x 4. 3 x 4.6 5 x 4.6 1 x 4.6 15 x 4.6 25 x 4.6 4 x 3.* /1pk C18 M-4439-D A-4439-E B-4439-E D-4439-E F-4439-E G-4439-E AJ-7597 C6-Phenyl A-4443-E B-4443-E D-4443-E F-4443-E G-4443-E AJ-7915 /1pk NX-C18 B-4453-E D-4453-E F-4453-E G-4453-E AJ-8368 for ID: 3.2-8. mm 5 µm Minibore and Narrow Bore Columns (mm) SecurityGuard Cartridges (mm) Phases 3 x 2. 5 x 2. 15 x 2. 25 x 2. 5 x 3. 1 x 3. 15 x 3. 25 x 3. 4 x 2.* /1pk C18 A-4435-B B-4435-B F-4435-B G-4435-B B-4435-Y D-4435-Y F-4435-Y G-4435-Y AJ-7596 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-4454-B B-4454-B F-4454-B B-4454-Y D-4454-Y F-4454-Y G-4454-Y AJ-8367 for ID: 2.-3. mm 5 µm Analytical Columns (mm) SecurityGuard Cartridges (mm) Phases 3 x 4.6 5 x 4.6 1 x 4.6 15 x 4.6 25 x 4.6 4 x 3.* /1pk C18 A-4435-E B-4435-E D-4435-E F-4435-E G-4435-E AJ-7597 C6-Phenyl A-4444-E B-4444-E D-4444-E F-4444-E G-4444-E AJ-7915 /1pk NX-C18 B-4454-E D-4454-E F-4454-E G-4454-E AJ-8368 for ID: 3.2-8. 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 45 days for a FULL REFUND. 17

Impact Rapid protein precipitation Quickly clean up sample by passing biological samples through the Impact filter Increase sensitivity of your analysis by eliminating proteins which contribute to baseline noise and decreased column lifetime Increase reproducibility with the leak-free membrane, preventing premature sample breakthrough and incomplete protein precipitation Impact Ordering Information Impact Precipitation Products Part No. Description Unit Impact Precipitation Products CE-7565 Impact Protein Precipitation, Square Well, Filter Plate, 2 ml 2/box Impact Starter Kit for Protein Precipitation CE-821 Impact Protein Precipitation Plate (2 ea) Collection Plate 2 ml (2 ea) Sealing Mat, Santoprene (AH-8199) (2 ea) ea If Impact does not perform as well or better than your current protein precipitation plate with similar specifications, return the product with comparative data within 45 days for a FULL REFUND. Product Limitations 18 The products offered in this catalog and Phenomenex Analyte Specific Reagent products are not intended for clinical use. Because they are not intended for clinical use, no claim or representation is made or intended for their clinical use (including, but not limited to diagnostic, prognostic, therapeutic or blood banking). It is the user s responsibility to validate the performance of Phenomenex products for any particular use, since the performance characteristics are not established. Phenomenex products may be used in clinical diagnostic laboratory systems after the laboratory has validated their complete system as required by the Clinical Laboratory Improvements Amendments of 1988 (CLIA 88) regulation in the U.S. or equivalent in other countries.

Phree Phree is a high-throughput solution for simultaneous removal of protein and all classes of phospholipids to maximize sensitivity and column lifetime. Phospholipid Removal Solutions Remove Proteins Solvent Shielding Technology prevents dripping of organic solvent, allowing for protein precipitation within the wells of the Phree Phospholipid Removal Product. Eliminate Phospholipids The Phree sorbent selectively removes phospholipids from precipitated plasma samples. No Method Development One method for acids, bases, and neutrals Proteins Phospholipids Target Analyte Phree Ordering Information Part No. Description Unit 8E-S133-TGB Phree Phospholipid Removal 96-Well Plates 2/box Collection Plates (deep well, polypropylene) AH-7192 Strata 96-Well Collection Plate 35 µl/well 5/pk AH-7193 Strata 96-Well Collection Plate 1 ml/well 5/pk AH-7194 Strata 96-Well Collection Plate 2 ml/well 5/pk AH-8635 Strata 96-Well Collection Plate, 2 ml Square/Round-Conical 5/pk AH-8636 Strata 96-Well Collection Plate, 2 ml Round/Round, 8 mm 5/pk AH-7279 Strata 96-Well Collection Plate, 1 ml/well Round, 7 mm 5/pk Sealing Mats AH-8597 Sealing Mats, Pierceable, 96-Square Well, Silicone 5/pk AH-8598 Sealing Mats, Pre-Slit, 96-Square Well, Silicone 5/pk AH-8631 Sealing Mats, Pierceable, 96-Round Well 7 mm, Silicone 5/pk AH-8632 Sealing Mats, Pre-Slit, 96-Round Well 7 mm, Silicone 5/pk AH-8633 Sealing Mats, Pierceable, 96-Round Well 8 mm, Silicone 5/pk AH-8634 Sealing Mats, Pre-Slit, 96-Round Well 8 mm, Silicone 5/pk AH-7362 Sealing Tape Pad 1/pk Vacuum Manifold AH-895 Strata 96-Well Plate Manifold, Universal with Vacuum Gauge ea If Phree Phospholipid Removal products do not perform as well or better than your current phospholipid removal products, return the product with comparative data within 45 days for a FULL REFUND. 19

) ) ) Solid Phase Extraction for Optimal Cleanup Strata-X U.S. Patent No. 7,119,145 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) 85 Surface Area (m 2 /g) 8 ph Stability 1-14 3 Mechanisms of Retention π-π Bonding Hydrogen Bonding Dipole-Dipole Interactions Hydrophobic Interaction )n O )n O )n O Ṅ. Ṅ. Ṅ. Strata-X Ordering Information Sorbent Format Mass Part Number Unit Tube 3 mg 8B-S1-TAK 1 ml (1/box) 3 mg 8B-S1-TBJ 3 ml (5/box) 6 mg 8B-S1-UBJ 3 ml (5/box) 1 mg 8B-S1-EBJ 3 ml (5/box) 1 mg 8B-S1-ECH 6 ml (3/box) 2 mg 8B-S1-FBJ 3 ml (5/box) 2 mg 8B-S1-FCH 6 ml (3/box) 5 mg 8B-S1-HBJ 3 ml (5/box) 5 mg 8B-S1-HCH 6 ml (3/box) Giga Tube 5 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) 5 g 8B-S1-LFF 6 ml (16/box) Teflon Tube 2 mg 8B-S1-FBJ-T 3 ml (5/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 Extraction Protocol 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 5-6 % Methanol in DI Water 4. Dry: Sorbent for 3 seconds 5. Elute: 2x 5 µl 2 % Formic acid in Methanol or Acetonitrile On-line Extraction Cartridge Description Part Number Unit/Box Strata-X on-line extraction M-S33-B-CB ea cartridge, 2 x 2. mm Cartridge holder, 2 mm CH-5845 ea 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 45 days for a FULL REFUND. Instant Method Development Create your Own SPE Method in under 1 minute! www.phenomenex.com/tools/mdtool 2

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. Email: phenologix@phenomenex.com or Visit: www.phenomenex.com/phenologix How can we be of service? PhenoLogix worldwide technical teams are here to provide full service method support and training for you and your lab. Our services include: Method Improvement and Optimization New Method Development Validation and Pre-validation Services On-Site Training and Consulting Partial list of resources used in the PhenoLogix lab Agilent, Shimadzu, ACQUITY and JASCO HPLC and UHPLC systems API 3 LC/MS/MS API 4 LC/MS/MS API 5 LC/MS/MS 4 Q-TRAP LC/MS/MS Waters Micromass Quattro MS Agilent and Shimadzu GC/MS systems Gilson and PerkinElmer liquid handlers

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