vitamin - phenomenexphx.phenomenex.com/lib/br98401112_w.pdf · 4 phenomenex l web: vitamin b 1 from...

VitaminTesting

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PLIC

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2Phenomenex l WEB: www.phenomenex.com

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

For Research Use Only. Not for use in diagnostic procedures.© 2013 Phenomenex Inc. All rights reserved.


Applications

Vitamin B1 from Whole Blood: Thiamine Diphosphate (TDP) ................................... 4-5

Vitamin B1 from Plasma: Thiamine Monophosphate (TMP) and Thiamine ............... 6-7

Vitamin B3 ................................................................................................................ 8-9

Vitamin B6 ............................................................................................................ 10-11

25-OH-Vitamin D2 and D3 .................................................................................... 12-13

25-OH-Vitamin D3 and 3-epi-25-OH-Vitamin D3 .................................................. 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) ....................................................20

Technical Support

PhenoLogixSM: Your Analytical Support Laboratory ...................................................21

Improve Your Vitamin Analysis


Vitamin B1 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 be-cause approximately 90 % of total thiamine in blood is in the red blood cells.Deficiency can lead to diseases such as Beriberi and optic neuropathy.

Method Highlights

• Total run time under 4 min

• Analytical measurement range: 0–400 nmol/L

• Good linearity: R2 = 0.9991

SAmPLE PrEPArATION

1. Collect: venous blood into EDTA or heparin containing tubes.

2. Freeze: sample immediately at -70 °C or -20 °C for up to 7 days.

3. Add: 500 µL of 10 % TCA to 500 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: 14,000 rpm for 6 minutes at room temperature. Note: The relative centrifugal force (RCF) = 16,000 g.

6. Transfer: 500 µL supernatant into a glass tube.

i. Wash twice with methyl-tert-butyl ether (750 µL) to remove TCA.

ii. Vortex for 15 seconds.

iii. Centrifuge the tubes at 3,500 rpm for 3 minutes.

iv. Remove the tubes and discard the top organic layer.

v. Repeat once.

7. Transfer: 50 µL of extract to an autosampler injection vial.

8. Add: 50 µL of water.

9. Add: 50 µL of derivatizing reagent.

10. Vortex: 15 seconds and place the vial on the autosampler at RT.

11. Inject: 20 µL of prepared sample.

*Derivatizing reagent: Potassium ferricyanide [K3Fe(CN)6] was prepared as the following: Dissolve 6 mg of K3Fe(CN)6 in 15 mL 15 % sodium hydroxide solution. Prepare fresh daily.

OHOH

OH

NH2

N

N

N

OO

OOS

PP

+

Vitamin B1

Thiamine Diphosphate


90

80

70

60

50

40

30

20

10

0

0 50 100 150 200 250

TDP Concentration (nmol/L)

300 350 400 450

y = 0.1969x + 0.6829R2 = 0.9991

Pea

k ar

ea

LC/mS/mS mETHOD

Multi-standards of TDP (0-400 nmol/L) injected after sample cleanup

min3.51.510.5

52.5

55

57.5

60

65

67.5

70

20877

mAU

0 3.5

Column: Gemini® 5 µm C18Dimensions: 50 x 4.6 mm

Part No.: 00B-4435-B0Mobile Phase: A: 25 mM Disodium hydrogen phosphate, 10 % Methanol, pH 7.0

B: 25 mM Disodium hydrogen phosphate, 70 % Methanol, pH 7.0Gradient: Time (min) % B

011.52.52.64.0

012.5509000

Flow Rate: 1.0 mL/minDetection: Fluorescence.

Excitation: 375 nm; Emission: 435 nmSample: TDP (thiamine diphosphate)

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 cleanupSeq. No. RT Area Peak HeightMean 2.634 66.8 12.0STDV 0.0052 0.3521 0.0823CV (%) 0.20 0.53 0.68

App

ID 2

0877

App

ID 2

0880

Standard curve of TDP (Whole blood), 0 – 400 nmol/L

Questions or requests?Contact [email protected] for questions on applications or to request additional applications.

Vitamin B1 from Whole Blood: Thiamine Diphosphate (TDP) (cont’d)


Vitamin B1 from Plasma: Thiamine monophosphate (TmP) and ThiamineTotal Thiamine reflects the measurement of Thiamine (Vitamin B1) and Thiamine Monophosphate (TMP).

Method Highlights

• Excellent linearity. TMP R2 = 0.9996. Thiamine R2 = 0.9986

SAmPLE PrEPArATION

1. Add: 400 µL of methanol to a well of an Impact Protein Precipitation Plate.

2. Add: 100 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: 50 µL to an autosampler vial (or leave sample in collection plate).

6. Add: 100 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.

All conditions are at room temperature (RT).

*Derivatizing reagent: Potassium ferricyanide [K3Fe(CN)6] was prepared as the following:

Dissolve 6 mg of K3Fe(CN)6 in 15 mL 15 % sodium hydroxide solution. Prepare fresh daily.

LC/mS/mS mETHOD

0 2 4 min

54

56

58

60

mAU

20875

12

App

ID 2

0875

N

N

NH2

N

S OOH

OHO

P

Vitamin B1

Thiamine Monophosphate

Column: Kinetex® 5 µm C18Dimensions: 100 x 4.6 mm

Part No.: 00D-4601-E0Mobile Phase: A: 25 mM Disodium hydrogen phosphate, 10 % Methanol, pH 7.0

B: 25 mM Disodium hydrogen phosphate, 70 % Methanol, pH 7.0Gradient: Time (min) % B

00.250.753.004.004.505.00

32525356033

Flow Rate: 1 mL/minTemperature: 25 °C

Backpressure: 150 barDetection: Fluorescence Detector

Excitation: 375 nm; Emission: 435 nmSample: 1. TMP (Thiamine monophospate)

2. Thiamine

200 nmol/L filtered by an Impact Protein Precipitation Plate


DATA SUmmAry

Plasma spiked with TMP Plasma spiked with ThiamineSeq. No. RT Area RT AreaMean 2.602 10.125 4.275 1.650STDV 0.0148 0.0707 0.0033 0.0756CV (%) 0.57 0.70 0.08 4.58

0 1 2 3 4 min

54

55

56

mAU

20881

12

5 nmol/L filtered by an Impact™ Protein Precipitation Plate

Standard Curve of TMP Filtered by an Impact Protein Precipitation Plate

App

ID 2

0881

35

30

25

20

15

10

5

00 50 100

TMP Concentration (nmoL/L)

150 200 250

Pea

k ar

ea

App

ID 2

0876


y = 0.1434x - 0.0306

R2 = 0.9996

Vitamin B1 from Plasma: Thiamine monophosphate (TmP) and Thiamine (cont’d)


Vitamin B3

Vitamin B3, also known as Niacin or Nicotinic Acid is an important part of the human diet. A deficiency of Vitamin B3 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

SAmPLE PrEPArATION

1. Place: Impact plate onto a suitable 96-well sample manifold or rotor.

2. Dispense: 300 µL acetonitrile into the wells of the Impact plate.

3. Add: 100 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

Vitamin B3

N

OH

O

0.50 1

1

1.5 2

2

2.5 3 3.5 min0

2.0e4

4.0e4

6e4

8e4

1e5

1.2e5

1.4e5

1.6e5

1.8e5

2e5

2.2e5

2.4e5

2.6e5

2.8e5

3e5

3.2e5

3.4e5

3.6e5

8290

Inte

nsity

, cp

s

App

ID 2

0909


Part No.: 00D-4439-E0Mobile Phase: A: 0.1 % Formic acid in water

B: MethanolGradient: Time (min)

02.52.64.0

% B10901010

Flow Rate: 0.6 mL/minDetection: Electrospray Mass Spec (ESMS)Detector: AB SCIEX API 4000™

Sample: 1. Nicotinamide2. Nicotinic Acid

LC/MS/MS Chromatogram of Nicotinamide and Nicotinic Acid at 1000 ng/mL


0.50 1.0

1

2

1.5 2.0 2.5 3.0 3.5 min0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000

7500

8000

20926

Inte

nsity

, cp

s

0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 minConcentration, ng/mL

0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

7.0e5

8.0e5

9.0e5

1.0e6

1.1e6

1.2e6

1.3e6

1.4e61.5e6

Are

a, c

ount

s

R2 = 0.9996

App

ID 2

0910

App

ID 2

0906

MRM Transitions Used for Data AnalysisPeak Name MRM ChannelNicotinamide 123.005 — 80.100Nicotinic acid 123.981 — 80.100


Part No.: 00D-4439-E0Mobile Phase: A: 0.1 % Formic acid in water

B: MethanolGradient: Time (min)

02.52.64.0

% B10901010

Flow Rate: 0.6 mL/minDetection: Electrospray Mass Spec (ESMS)Detector: AB SCIEX API 4000™

Sample: 1. Nicotinamide2. Nicotinic Acid

Nicotinic Acid Standard Curve, 2- 2000 ng/mL

LC/MS/MS Chromatogram of Nicotinamide and Nicotinic Acid at 10 ng/mL

Vitamin B3 (cont’d)


Vitamin B6

Vitamin B6 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 com-pounds, PLP is the primary biologically active form of vitamin B6.

Method Highlights

• 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 400 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 30 μL of 250 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 30 minutes.

5. Uncap the tubes; add 50 μL of 20 % meta-Phosphoric acid to the controls and patient samples.

6. Recap the tubes and vortex for 30 seconds at room temperature.

7. Centrifuge for 5 minutes at 14,000 rpm. Note: The relative centrifugal force (RCF)=16,000 g.

8. Transfer 350 μL of supernatant to a 2 mL amber vial.

9. Cover the vial with lid and place in the autosampler (RT).

10. Inject 30 µL.

HO

O

O O

HO OH

N

P

Vitamin B6


Vitamin B6 (cont’d)

HPLC ANALySIS

Sample chromatogram of semicarbazide derivatized PLP and PL

y = 65.442x - 154.18

R2 = 0.9999

0

2000

4000

6000

8000

10000

12000

14000

0 50 100 150 200 250

PLP Concentration in Human Serum (nmol/L)

Pea

k A

rea

HUmAN SErUm PLP STANDArD CUrVE

Example Standard Curve from 6.25 nmol/L to 200 nmol/L for PLP in Human Serum (external standard method)

10 2 3 4 5 6 min

mAu

5500

5750

6000

6250

6500

6750

7000

7250

75001

2

3Column: Gemini® NX-C18 3 µm

Dimensions: 100 x 4.6 mmPart No.: 00D-4453-E0

Guard: SecurityGuard™ Cartridge C18, 4 x 3.0 mm IDPart No.: AJ0-8368

Mobile Phase: A: 20 mM Sodium phosphate and 1.0 mL Acetic acid in DI water; pH = ~6.0; no pH adjustment.B: Acetonitrile/Methanol (70:30)

Gradient: Time (min)05.05.16.06.17.0

% B560959555

Flow Rate: 1 mL/minInjection Volume: 30 µL

Temperature: 35 °CDetection: Fluorescence: Ex 360; Em 450

Sample: 1. PLP (Pyridoxal 5’-Phosphate)2. PA (4-pyridoxic acid)3. PL (Pyridoxal)

App

ID 2

0684

App

ID 2

0922



25-OH-Vitamin D2 and D3

Vitamin D is recognized as an essential nutrient for intestinal absorption of calcium and phosphate and for promoting bone resorption and formation. De-ficiency 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 D2 and 25-OH D3.

Method Highlights

• Customer-submitted patient sample run on a multi-channel system

• 40-50 µL injection volume

SAmPLE PrEPArATION

1. Add: 50 µL of precipitating reagent containing internal standard to a 1.5 mL centrifuge tube.

2. Pipette: 100 µL of serum into a centrifuge tube.

3. Vortex: 20 – 30 seconds.

4. Inspect: each tube to ensure no unmixed sample remains in the bottom of the tube.

i. A homogeneous mixture is critical.

ii. If unmixed sample remains at the bottom of the tube, dislodge by invert-ing and tapping, then re-vortex.

5. Centrifuge: 15 minutes at 13,000 rpm.

6. Transfer: supernatant into sample vial without disturbing the pellet.

Vitamin D3 – cholecalciferol

Vitamin D2 – ergocalciferol

HO

H

TIP:Consider Impact™ for high-throughput protein precipitation. See www.phenomenex.com/impact


25-OH-Vitamin D2 and D3 (cont’d)

LC/MS/MS AnALySiSAnalyte Q1 Q325-OH D2 395.3 209.325-OH D3 383.2 257.225-OH D3-d6 389.3 263.3

19314

6.0e4

5.0e4

4.0e4

3.0e4

2.0e4

1.0e4

0

1.14e4

1.00e4

8000

6000

4000

2000

0

1.6e4

1.4e4

1.2e4

1.0e4

8000

6000

4000

2000

0

3

1

2

Inte

nsity

, cp

sIn

tens

ity, c

ps

Inte

nsity

, cp

s

25-OH D3-d6 (IS)

25-OH D2

25-OH D3

3.6 3.7 3.8 3.9 4.0 4.1 4.2 4.3 4.4 4.5 4.6 min

3.6 3.7 3.8 3.9 4.0 4.1 4.2 4.3 4.4 4.5 4.6 min

3.6 3.7 3.8 3.9 4.0 4.1 4.2 4.3 4.4 4.5 4.6 min

Patient sample run on a multi-channel system

Column: Kinetex® 2.6 µm C18Dimensions: 50 x 4.6 mm

Part No.: 00B-4462-E0Mobile Phase: A: 0.05 % Formic acid in Water

B: 5 mM Ammonium acetate with 0.1 % Formic acid in MethanolGradient: Time (min)

00.123.44.86

% B881001008

Flow Rate: 1 mL/minTemperature 35 ºC

Detection: Mass Spectrometer (MS)Detector: AB SCIEX API 4000™

Sample: 1. 25-Hydroxyvitamin D2 (25-OH D2) 2. 25-Hydroxyvitamin D3 (25-OH D3)3. 25-Hydroxyvitamin D3-d6 (25-OH D3-d6)

App

ID 1

9314


View our technical note on 25-OH-Vitamin D2 and D3 at www.phenomenex.com/clinical


25-OH-Vitamin D3 and 3-epi-25-OH-Vitamin D3

Vitamin D (Ergocalciferol, D2 and Cholecalciferol, D3) 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 (conver-sion 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 D3 and 3-epi-25-OH-Vitamin D3 for accurate quantitation using Kinetex® 2.6 µm PFP HPLC column

• Fast run time in less than 5 minutes

LC/mS/mS ANALySISColumn: Kinetex 2.6 µm PFP

Dimensions: 100 x 2.1 mmPart No.: 00D-4477-AN

Mobile Phase: A: 0.1 % Formic acid in DI WaterB: 0.1 % Formic acid in Methanol

Gradient: Time (min)02.003.803.816.00

% B7580807575

Flow Rate: 0.4 mL/minTemperature: Ambient

Detection: Mass Spectrometer (MS/MS)Detector: AB SCIEX API 5000™

mrm TrANSITIONSAnalyte Q1 Q3OH-Vit D2 395.3 209.3OH-Vit D3/Epi-D3 383.2 257.2IS (OH-D3-2H3) 386.2 257.2OH-Vit D3 (Sec trans) 383.2 229.1OH-Vit D2 (Sec trans) 395.3 269.2

View our technical note on 3-epi-25-OH Vitamin D3 at www.phenomenex.com/clinical


00

2.0e5

4.0e5

6.0e5

8.0e5

1.0e6

1.2e6

1.4e6

1.6e6

Inte

nsity

, cp

s

1.8e6

2.0e6

2.2e6

2.4e6

0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 min

5 10

0.34

0.32

0.28

0.30

0.260.24

0.22

0.20

0.180.16

0.140.12

0.100.08

0.06

0.04

0.02

0.0015 20 25 30 35 40 45 50 55 60 5 70 75 80 85 90 95 100

Analyte Conc. / IS Conc.

Ana

lyte

Are

a /

IS A

rea

5 10

1.20

1.10

1.00

0.90

0.80

0.70

0.60

0.50

0.40

0.30

0.20

0.10

0.0015 20 25 30 35 40 45 50 55 60 5 70 75 80 85 90 95 100

Analyte Conc. / IS Conc.

Ana

lyte

Are

a /

IS A

rea

25-OH-Vitamin D3 and 3-epi-25-OH-Vitamin D3 (cont’d)

25-OH-D23-Epi-25-OH-D3

25-OH-D3

App

ID 2

0007

App

ID 2

0006

OH-Vitamin D2 Calibration Curve from 2.5 – 100 ng/mL

OH-Vitamin D3 Calibration Curve from 2.5 – 100 ng/mL

App

ID 2

0003


AQ0-8503

UHPLC / HPLC SURe-Lok™ HigH PReSSURe Peek MALe nUt FittingSPart No. Description UnitAQ0-8503 Sure-Lok High Pressure PEEK 1-Pc Nut 10-32,

for 1/16 in. Tubing, 12,000 psi (827 bar) 10/pk

AQ0-8530 Sure-Lok Fitting Tightening Tool, Aluminum eaAQ0-8530

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 20,000 psi (1,378 bar)

• Fits virtually all manufacturers’ columns

SECUrITyGUArD ULTrA CArTrIDGE HOLDErSPart No. Description UnitAJ0-9000 SecurityGuard ULTRA

Cartridge Holderea

‡SecurityGuard ULTRA cartridges require holder, Part No.: AJ0-9000

2.6 µm MidBore™ Columns (mm) SecurityGuard ULTRA Cartridges‡

Phases 30 x 3.0 50 x 3.0 75 x 3.0 100 x 3.0 150 x 3.0 3/pk

XB-C18 00A-4496-Y0 00B-4496-Y0 00C-4496-Y0 00D-4496-Y0 00F-4496-Y0 AJ0-8775C18 00A-4462-Y0 00B-4462-Y0 00C-4462-Y0 00D-4462-Y0 00F-4462-Y0 AJ0-8775C8 00A-4497-Y0 00B-4497-Y0 00C-4497-Y0 00D-4497-Y0 00F-4497-Y0 AJ0-8777PFP 00A-4477-Y0 00B-4477-Y0 00C-4477-Y0 00D-4477-Y0 00F-4477-Y0 AJ0-8780HILIC 00A-4461-Y0 — — — 00F-4461-Y0 AJ0-8779Phenyl-Hexyl — — — 00D-4495-Y0 00F-4495-Y0 AJ0-8781

for 3.0 mm ID

Ordering Information

2.6 µm Analytical Columns (mm) SecurityGuard™ ULTRA Cartridges‡

Phases 30 x 4.6 50 x 4.6 75 x 4.6 100 x 4.6 150 x 4.6 3/pk

XB-C18 — 00B-4496-E0 00C-4496-E0 00D-4496-E0 00F-4496-E0 AJ0-8768C18 00A-4462-E0 00B-4462-E0 00C-4462-E0 00D-4462-E0 00F-4462-E0 AJ0-8768C8 — 00B-4497-E0 00C-4497-E0 00D-4497-E0 00F-4497-E0 AJ0-8770PFP 00A-4477-E0 00B-4477-E0 00C-4477-E0 00D-4477-E0 00F-4477-E0 AJ0-8773HILIC 00A-4461-E0 00B-4461-E0 00C-4461-E0 00D-4461-E0 00F-4461-E0 AJ0-8772Phenyl-Hexyl — 00B-4495-E0 — 00D-4495-E0 00F-4495-E0 AJ0-8774

for 4.6 mm ID

1.7 µm Minibore Columns (mm) SecurityGuard ULTRA Cartridges‡

Phases 30 x 2.1 50 x 2.1 100 x 2.1 150 x 2.1 3/pk

XB-C18 00A-4498-AN 00B-4498-AN 00D-4498-AN 00F-4498-AN AJ0-8782C18 00A-4475-AN 00B-4475-AN 00D-4475-AN 00F-4475-AN AJ0-8782C8 00A-4499-AN 00B-4499-AN 00D-4499-AN 00F-4499-AN AJ0-8784PFP 00A-4476-AN 00B-4476-AN 00D-4476-AN 00F-4476-AN AJ0-8787HILIC 00A-4474-AN 00B-4474-AN 00D-4474-AN — AJ0-8786Phenyl-Hexyl — 00B-4500-AN 00D-4500-AN 00F-4500-AN AJ0-8788

for 2.1 mm ID

1.7 µm MidBore Columns (mm) SecurityGuard ULTRA Cartridges‡

Phases 30 x 3.0 50 x 3.0 100 x 3.0 3/pk

XB-C18 00A-4498-Y0 00B-4498-Y0 00D-4498-Y0 AJ0-8775C18 — 00B-4475-Y0 00D-4475-Y0 AJ0-8775C8 00A-4499-Y0 00B-4499-Y0 00D-4499-Y0 AJ0-8777PFP — — 00D-4476-Y0 AJ0-8780HILIC — 00B-4474-Y0 — AJ0-8779Phenyl-Hexyl — — — AJ0-8781

for 3.0 mm ID

2.6 µm Minibore Columns (mm) SecurityGuard ULTRA Cartridges‡

Phases 30 x 2.1 50 x 2.1 100 x 2.1 150 x 2.1 3/pk

XB-C18 00A-4496-AN 00B-4496-AN 00D-4496-AN 00F-4496-AN AJ0-8782C18 00A-4462-AN 00B-4462-AN 00D-4462-AN 00F-4462-AN AJ0-8782C8 00A-4497-AN 00B-4497-AN 00D-4497-AN 00F-4497-AN AJ0-8784PFP 00A-4477-AN 00B-4477-AN 00D-4477-AN 00F-4477-AN AJ0-8787HILIC 00A-4461-AN 00B-4461-AN 00D-4461-AN 00F-4461-AN AJ0-8786Phenyl-Hexyl 00A-4495-AN 00B-4495-AN 00D-4495-AN 00F-4495-AN AJ0-8788

for 2.1 mm ID

Kinetex®

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.

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/10pkC18 00M-4439-D0 00A-4439-E0 00B-4439-E0 00D-4439-E0 00F-4439-E0 00G-4439-E0 AJ0-7597C6-Phenyl — 00A-4443-E0 00B-4443-E0 00D-4443-E0 00F-4443-E0 00G-4443-E0 AJ0-7915

— — /10pk

NX-C18 — — 00B-4453-E0 00D-4453-E0 00F-4453-E0 00G-4453-E0 AJ0-8368for ID: 3.2-8.0 mm

5 µm Analytical Columns (mm) SecurityGuard Cartridges (mm)Phases 30 x 4.6 50 x 4.6 100 x 4.6 150 x 4.6 250 x 4.6 4 x 3.0*

/10pkC18 00A-4435-E0 00B-4435-E0 00D-4435-E0 00F-4435-E0 00G-4435-E0 AJ0-7597C6-Phenyl 00A-4444-E0 00B-4444-E0 00D-4444-E0 00F-4444-E0 00G-4444-E0 AJ0-7915

— /10pkNX-C18 — 00B-4454-E0 00D-4454-E0 00F-4454-E0 00G-4454-E0 AJ0-8368

for ID: 3.2-8.0 mm


3 µm Microbore, Minibore and Narrow Bore Columns (mm) SecurityGuard™ Cartridges (mm)Phases 50 x 1.0 20 x 2.0 30 x 2.0 50 x 2.0 100 x 2.0 150 x 2.0 50 x 3.0 100 x 3.0 150 x 3.0 4 x 2.0*

/10pkC18 00B-4439-A0 00M-4439-B0 00A-4439-B0 00B-4439-B0 00D-4439-B0 00F-4439-B0 00B-4439-Y0 00D-4439-Y0 00F-4439-Y0 AJ0-7596

C6-Phenyl 00B-4443-A0 — 00A-4443-B0 00B-4443-B0 00D-4443-B0 00F-4443-B0 00B-4443-Y0 00D-4443-Y0 00F-4443-Y0 AJ0-7914— /10pk

NX-C18 — 00M-4453-B0 00A-4453-B0 00B-4453-B0 00D-4453-B0 00F-4453-B0 00B-4453-Y0 00D-4453-Y0 00F-4453-Y0 AJ0-8367for ID: 2.0-3.0 mm

*SecurityGuard™ Analytical Cartridges require holder, Part No.: KJ0-4282

U.S. Patent No. 7,563,367

5 µm Minibore and Narrow Bore Columns (mm) SecurityGuard Cartridges (mm)Phases 30 x 2.0 50 x 2.0 150 x 2.0 250 x 2.0 50 x 3.0 100 x 3.0 150 x 3.0 250 x 3.0 4 x 2.0*

/10pkC18 00A-4435-B0 00B-4435-B0 00F-4435-B0 00G-4435-B0 00B-4435-Y0 00D-4435-Y0 00F-4435-Y0 00G-4435-Y0 AJ0-7596C6-Phenyl 00A-4444-B0 00B-4444-B0 00F-4444-B0 — 00B-4444-Y0 — 00F-4444-Y0 00G-4444-Y0 AJ0-7914

— /10pkNX-C18 00A-4454-B0 00B-4454-B0 00F-4454-B0 — 00B-4454-Y0 00D-4454-Y0 00F-4454-Y0 00G-4454-Y0 AJ0-8367

for ID: 2.0-3.0 mm

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.

Gemini®


ImpactRapid protein precipitation

Product Limitations

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.

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.

ImPACTOrdering InformationImpact Precipitation ProductsPart No. Description UnitImpact Precipitation ProductsCE0-7565 Impact Protein Precipitation, Square Well, Filter Plate, 2 mL 2/box

Impact Starter Kit for Protein PrecipitationCE0-8201 Impact Protein Precipitation Plate (2 ea)

Collection Plate 2 mL (2 ea) Sealing Mat, Santoprene™ (AH0-8199) (2 ea)

ea

• 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


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.

Phospholipid Removal Solutions

PHrEEOrdering Information Part No. Description Unit8E-S133-TGB Phree Phospholipid Removal 96-Well Plates 2/box Collection Plates (deep well, polypropylene)AH0-7192 Strata® 96-Well Collection Plate 350 µL/well 50/pkAH0-7193 Strata 96-Well Collection Plate 1 mL/well 50/pkAH0-7194 Strata 96-Well Collection Plate 2 mL/well 50/pkAH0-8635 Strata 96-Well Collection Plate, 2 mL Square/Round-Conical 50/pkAH0-8636 Strata 96-Well Collection Plate, 2 mL Round/Round, 8 mm 50/pkAH0-7279 Strata 96-Well Collection Plate, 1 mL/well Round, 7 mm 50/pkSealing MatsAH0-8597 Sealing Mats, Pierceable, 96-Square Well, Silicone 50/pkAH0-8598 Sealing Mats, Pre-Slit, 96-Square Well, Silicone 50/pkAH0-8631 Sealing Mats, Pierceable, 96-Round Well 7 mm, Silicone 50/pkAH0-8632 Sealing Mats, Pre-Slit, 96-Round Well 7 mm, Silicone 50/pkAH0-8633 Sealing Mats, Pierceable, 96-Round Well 8 mm, Silicone 50/pkAH0-8634 Sealing Mats, Pre-Slit, 96-Round Well 8 mm, Silicone 50/pkAH0-7362 Sealing Tape Pad 10/pkVacuum ManifoldAH0-8950 Strata 96-Well Plate Manifold, Universal with Vacuum Gauge ea

PhreePhree is a high-throughput solution for simultaneous removal of protein and all classes of phospholipids to maximize sensi-tivity and column lifetime.

remove ProteinsSolvent Shielding Technology™ prevents drip-ping of organic solvent, allowing for protein precipitation within the wells of the Phree Phospholipid Removal Product.

Eliminate PhospholipidsThe Phree sorbent selectively removes phospholipids from precipitated plasma samples.

Proteins Phospholipids target Analyte

No method DevelopmentOne method for acids, bases, and neutrals


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 DevelopmentCreate your Own SPE Method in under 1 minute! www.phenomenex.com/tools/MDtool

Solid Phase Extraction for Optimal Cleanup

Strata-XU.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.

GENErAL EXTrACTION PrOTOCOL

Method written for a 30 mg/1 mL tube

1. Condition 1 mL Methanol followed by 1 mL DI Water

2. Load: Pretreated sample

3. Wash: 1 mL 5-60 % Methanol in DI Water

4. Dry: Sorbent for 30 seconds

5. Elute: 2x 500 µL 2 % Formic acid in Methanol or Acetonitrile


FormatSorbent

Mass Part Number UnitTube

30 mg 8B-S100-TAK 1 mL (100/box)30 mg 8B-S100-TBJ 3 mL (50/box) 60 mg 8B-S100-UBJ 3 mL (50/box)

100 mg 8B-S100-EBJ 3 mL (50/box) 100 mg 8B-S100-ECH 6 mL (30/box) 200 mg 8B-S100-FBJ 3 mL (50/box) 200 mg 8B-S100-FCH 6 mL (30/box) 500 mg 8B-S100-HBJ 3 mL (50/box) 500 mg 8B-S100-HCH 6 mL (30/box)

Giga™ Tube500 mg 8B-S100-HDG 12 mL (20/box)

1 g 8B-S100-JDG 12 mL (20/box)1 g 8B-S100-JEG 20 mL (20/box)2 g 8B-S100-KEG 20 mL (20/box)5 g 8B-S100-LFF 60 mL (16/box)

Teflon® Tube200 mg 8B-S100-FBJ-T 3 mL (50/box)

200 mg 8B-S100-FDG-T 12 mL (20/box)

96-Well Plate10 mg 8E-S100-AGB 2 Plates/Box30 mg 8E-S100-TGB 2 Plates/Box60 mg 8E-S100-UGB 2 Plates/Box

On-line Extraction Cartridge Description Part Number Unit/Box

Strata-X on-line extraction cartridge, 20 x 2.0 mm

00M-S033-B0-CB ea

Cartridge holder, 20 mm CH0-5845 ea

STrATA-X

3 Mechanisms of Retention

Hydrogen Bonding Dipole-Dipole Interactions

N..

n)

)

O

π-π Bonding

N..

n)

)

O

Hydrophobic Interaction

N..

n)

)

O

Material CharacteristicsParticle Size (µm) 33Pore Size (Å) 85Surface Area (m2/g) 800pH Stability 1-14

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 3000™ LC/MS/MS



• 4000 Q-TRAP® LC/MS/MS

• Waters® Micromass® Quattro™ MS

• Agilent and Shimadzu GC/MS systems

• Gilson® and PerkinElmer® liquid handlers

An Analytical Support LaboratoryPhenoLogix 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: [email protected] orVisit: www.phenomenex.com/Phenologix

www.phenomenex.comPhenomenex products are available worldwide. For the distributor in your country, contact Phenomenex USA, International Department at [email protected]

t: f:

Australia02-9428-6444 02-9428-6445 [email protected]

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Austria 01-319-1301 01-319-1300 [email protected]

t: t: f:

Belgium02 503 4015 (French) 02 511 8666 (Dutch) +31 (0)30-2383749 [email protected]

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Canada(800) 543-3681 (310) 328-7768 [email protected]

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Denmark4824 8048 +45 4810 6265 [email protected]

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Finland09 4789 0063 +45 4810 6265 [email protected]

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France01 30 09 21 10 01 30 09 21 11 [email protected]

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germany 06021-58830-0 06021-58830-11 [email protected]

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india 040-3012 2400 040-3012 2411 [email protected]

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ireland 01 247 5405 +44 1625-501796 [email protected]

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italy 051 6327511 051 6327555 [email protected]

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Luxembourg+31 (0)30-2418700 +31 (0)30-2383749 [email protected]

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Mexico001-800-844-5226 001-310-328-7768 [email protected]

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the netherlands030-2418700 030-2383749 [email protected]

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new Zealand09-4780951 09-4780952 [email protected]

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norway 810 02 005 +45 4810 6265 [email protected]

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Puerto Rico (800) 541-HPLC (310) 328-7768 [email protected]

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Sweden 08 611 6950 +45 4810 6265 [email protected]

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United kingdom 01625-501367 01625-501796 [email protected]

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United States(310) 212-0555 (310) 328-7768 [email protected]

t: f:

All other countries: Corporate office USA (310) 212-0555 (310) 328-7768 [email protected]

BR

9840

1112

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Terms and Conditions Subject to Phenomenex Standard Terms & Conditions, which may be viewed at www.phenomenex.com/TermsAndConditions.

Trademarks Phenomenex, Luna, Gemini, Kinetex, and Strata are registered trademarks, and MidBore, Phree, Solvent Shielding Technology, pH-LC, Impact, Giga, SecurityGuard, Strata-X, Sure-Lok, Synergi, and TWIN are trademarks of Phenomenex. Micromass, Waters, and ACQUITY are registered trademarks, and Quattro is a trademark of Waters Corporation. JASCO is a registered trademark of JASCO, Inc. Agilent is a registered trademark of Agilent Technologies, Inc. API 3000, API 4000, and API 5000 are trademarks and QTRAP is a registered trademark of AB SCIEX Pte. Ltd. Gilson is a registered trademark of Gilson, Inc. PerkinElmer is a registered trademark of PerkinElmer Inc. SGE is a registered trademark and FocusLiner is a trademark of SGE. Teflon is a registered trademark of E.I. du Pont de Nemours. Shimadzu is a registered trademark of Shimadzu Corporation.

Disclaimer Phenomenex is not affiliated with Agilent Technologies, JASCO, Gilson, PerkinElmer, or Waters Corpora-tion. Comparative separations may not be representative of all applications.

Gemini-NX is patented by Phenomenex. U.S. Patent No. 7,563,367

SecurityGuard is patented by Phenomenex. U.S. Patent No. 6,162,362

CAUTION: this patent only applies to the analytical-sized guard cartridge holder, and does not apply to SemiPrep, PREP or ULTRA holders, or to any cartridges.

Strata-X is patented by Phenomenex. U.S. Patent No. 7,119,145

© 2013 Phenomenex, Inc. All rights reserved.

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