Figure 3
A small amount ofmethanol finely tunes the separationof narcotic analgesics and acetaminophen
on anUltraC18 column, as indicatedby lesspeak tailing.
• 7 •
800-356-1688
These changes led to a slight increase in tailing for
all compounds on both Ultra C8 and Pinnacle II
™
C8 columns, but this was acceptable, especially
because the run time for the analysis was reduced
by a factor of 3 and resolutionwas improved by
59% to 79%. The system passed the system suitabil-
ity requirements in the USPmonograph.
In the next experiment, we re-introduced the ion
pair reagent hexane sulfonic acid into the system
under the control of the pH 2.5 phosphate buffer
system. The run time doubled, relative to the origi-
nal procedure, demonstrating that TEA did affect
the concentration of the ion-pairing agent.
Reducing the concentration of ion pairing agent, or
using a shorter chain length ion-pairing agent,
might have been a better alternative to adding TEA.
The system still passed the system suitability
requirements listed by the USP, but the chro-
matogramwasmuch noisier—and equilibration
problems seen in the USP 25 analysis returned.
After reviewing themonographs for admixtures con-
taining structurally related narcotics and acetamino-
phen, we created a single separation formorphine
sulfate, acetaminophen, codeine phosphate, oxy-
codoneHCl, and hydrocodone bitartrate. The goal
was to create an adequate separationwhile keeping
themethod as simple as possible. We chose anUltra
C18 column and set detection to 235nm. All compo-
nents, including a small unknown peak, were sepa-
rated to baseline (Figure 2).
Next, we increased the amount of buffer to 90% (a
5% increase). This simple increase doubled the
analysis time. Resolution doubled betweenmost
components, with the greatest change between acet-
aminophen and codeine. The unknown peak disap-
peared and probably co-elutedwithmorphine.
We adjusted themobile phase ratio to 85:15,
buffer:organic solvent, using a 90:10mixture of
acetonitrile andmethanol as the organic solvent.
Resolution improved, relative to the original mobile
phase composition, analysis againwas under 10
minutes, and the unknown peak returned (Figure
3). For this analysis, these conditions provided the
most desirable results.
In summary, the goal of anymethod should be to
achieve themost stable and robust separation.
Sometimesmethods aremore complicated than they
need to be, and this canmake analysis unnecessari-
ly difficult. Even troubleshooting suchmethods adds
to production costs. When preparing to follow a
method always attempt to determine the reason a
reagent would be included in amobile phase. Any
change ormodification should have an established
scientific purpose. By creatingmore universal meth-
ods for analyses of structurally related compounds,
it should be possible to reduce costs for supplies,
increase laboratory analysis efficiency, and reduce
personnel training time.
For chromatograms illustrating the changes in sep-
aration that occur with each change in themobile
phase, please request Applications Note #59453. If
you encounter problems when analyzing your sam-
ples according to an establishedmethod, our expe-
rienced Technical Service chemists will be glad to
help. Contact them at 800-356-1688, ext. 4 or 814-
353-1300, ext. 4, or contact your Restek represen-
tative.
Peak List:
Conc. (µg/mL)
Ret. Time (min.) Tailing Resolution
U. unknown
unknown
3.1
NA
NA
1.morphine sulfate
204
3.3
1.0
1.8
2. acetaminophen
92
5.0
1.1
14.1
3. codeine phosphate
216
5.5
1.4
2.6
4. oxycodone HCl
206
7.5
1.4
8.5
5. hydrocodone bitartrate 218
8.9
1.4
5.0
Sample:
Inj.:
4.0µL
Sample:
rawmaterial mix
Solvent:
mobile phase
LC_0218
Column:
Ultra C18
Catalog #:
9174575
Dimensions:
250 x 4.6mm
Particle size:
5µm
Pore size:
100Å
Conditions:
Mobile phase:
A: pH 2.8 10mm
potassium phosphate
B: acetonitrile:
methanol (90:10 v/v)
(85A:15B, v/v)
Flow:
1.0mL/min.
Temp.:
27°C
Det.:
UV@235nm
Ordering Information
| UltraC18 5µmColumns
1.0mm ID
2.1mm ID
3.2mm ID 4.0mm ID 4.6mm ID
Length
cat.#
cat.#
cat.#
cat.#
cat.#
30mm
9174531
9174532
9174533
— 9174535
50mm
9174551
9174552
9174553
— 9174555
100mm
9174511
9174512
9174513
9174514
9174515
150mm
9174561
9174562
9174563
9174564
9174565
200mm
9174521
9174522
9174523
— 9174525
250mm
9174571
9174572
9174573
— 9174575
Trident
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compare
values to
Figure 2
