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Pharmaceutical/Biotech
Applications
Nadifloxacin in Solution
Using 60mg 3mL HyperSep Retain-AX Extraction Column
(Part Number: 60107-403)
Sample Preparation
Prepare a 5ppm sample in 50mM phosphate buffer
pH 7.4
Condition Retain-AX Extraction Column
1mL of methanol
1mL of 2N NaOH
1mL of DI H
2
O
Apply Sample
Load 5mL samples at 1 to 2mL/minute
Wash Column
1mL of 5% ammonia aqueous solution
1mL of methanol
Elute Nadifloxacin
3mL of methanol with 4% acetic acid
Analysis
Mobile phase: ac
etonitrile:1mMKH
2
PO
4
(pH 3)
(40:60, v/v)
Flow: 1mL/minute
Temperature: 30°C
Detection: UV 254nm
Recommended HPLC Column
Part Number
Hypersil GOLD 5µm, 150 x 4.6mm
25005-154630
Nicotine and Continine in Urine or Serum
for GC or GC/MS Confirmations
Using 200mg 10mL HyperSep Verify-CX Extraction Column
(Part Number: 60108-742)
Prepare Sample
To 2mL of 100mM phosphate buffer (pH =6.0) add
internal standards*
Add 2mL of urine or serum
Sample pH should be 6.0±0.5
Adjust pH accordingly with 100mM monobasic or
dibasic sodium phosphate
Mix/vortex
Centrifuge as appropriate
Condition HyperSep Verify-CX Extraction Column
1 x 3mL CH
3
OH
1 x 3mL DI H
2
O
1 x 1mL 100mM phosphate buffer (pH 6.0)
NOTE:
Aspirate at <3"Hg to prevent sorbent drying
Apply Sample
Load at 1mL/minute
Wash Column
1 x 3mL DI H
2
O
1 x 2mL 200mM HCl
Dry column (5 minutes at >10"Hg)
1 x 2mL Hexane
Wash Column
Remove rack of collection tubes to rewash columns
1 x 3mL CH
3
OH
Dry column, (5 minutes at >10"Hg)
Elute Continine and Nicotine
Replace rack of collection tubes
1 x 3mL CH
2
Cl
2
/IPA/NH
4
OH (78:20:2)
Collect eluate at 1mL/minute
NOTE:
Prepare elution solvent fresh daily; add IPA/NH
4
OH mix,
then add CH
2
Cl
2
(pH 11-12)
Concentrate
Evaporate to dryness at <40°C
Take care not to over-heat or over evaporate
Reconstitute with 100µL ethyl acetate
Quantitate
Inject 1 to 2µL onto GC/MS
Monitor the following ions:
Compound
Primary Ion** Secondary Tertiary
Nicotine
84
133
162
Nicotine-D4*
88
137
166
Cotinine
98
119
176
Cotinine-D3*
101
122
179
* Suggested internal standard: Nicotine-D4, Cotinine-D3
** Quantitation Ion
Recommended GC Column
Part Number
TraceGOLD TG-5MS 30m x 0.25m x 0.25µm
26098-1420