PeakList:
Ret. Time
(min.)
%Inj. RSD (n=5)
1. hydrocodone bitartrate
3.1
0.37%
Sample:
Inj vol:
20µL
Conc.:
1mg/mL
Sample:
assay standard
Solvent:
mobile phase
Column:
Pinnacle II
™
Silica
Catalog #:
9210575
Dimensions: 250 x 4.6mm
Particle size: 5µm
Pore size:
110Å
Conditions:
Mobile phase: A: 800mL acetonitrile;
4mLwater; 1mL diethylamine
B: methanol
(55A:45B, v/v)
Flow:
1.5mL/min.
Temp.:
27°C
Det.:
UV@ 280nm
Restek Corporation • (800) 356-1688 • (814) 353-1300 •
sulfonic acid into the system under the control of the pH 2.5 phos-
phate buffer system. The run time doubled, 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 theUSP, but the chromatogramwasmuch noisier—and
equilibration problems returned (Application 3).
In addition to oxycodoneHCl rawmaterial, we chromatographed
hydrocodone bitartrate rawmaterial,morphine sulfate, and an
admixture of hydrocodone bitartrate and acetaminophen.Allmeth-
ods followedUSP 25 requirements and all chromatograms passed
system suitability requirements (Application 4).
After reviewing themonographs for admixtures containing struc-
turally related narcotics and acetaminophen, we created a single sep-
aration formorphine sulfate, acetaminophen, codeine phosphate,
oxycodoneHCl, and hydrocodone bitartrate. The goal was to create
an adequate separationwhile keeping themethod as simple as possi-
ble. For this purposewe chose anUltraC18 column and set detec-
tion to 235nm.All components, including a small unknown peak,
were separated to baseline (Application 5).
Next, we increased the amount of buffer to 90% (a 5% increase), to
determine the effect on the separation. The separationwas very sen-
sitive to the amount of buffer present; this simple 5% increase dou-
bled the analysis time. Resolution doubled betweenmost compo-
nents, with the greatest change between acetaminophen and
codeine. The unknown peak disappeared, however, and probably
co-elutedwithmorphine (Application 6).
To further improve resolution for codeine and the other later eluting
components, we adjusted themobile phase ratio to 85:15,
buffer:organic solvent, using a 90:10mixture of acetonitrile and
methanol as the organic solvent. Resolution improved, relative to
the originalmobile phase composition, analysis againwas under 10
minutes, and the co-eluting unknown peak returned (Application 7).
In an effort to further improve peak shape, we heated the column to
35°C. The higher temperature reduced the analysis time by one
minute, at the sacrifice of resolution between analytes, andwith fur-
ther distortion of the leading baseline for oxycodone and
hydrocodone (Application 8). For this analysis, the conditions used
to produce (Application 7) provided themost desirable results.
In summary, the goal of anymethod should be to achieve themost
stable and robust separation possible. Too oftenmethods aremade
more complicated than they need be, perhaps from lack of chro-
matographic experience or, possibly, tomake analysis unnecessarily
difficult. Even troubleshooting suchmethods adds to production
costs.When preparing to follow a compendiummethod always
Analyses of morphine sulfate and a hydrocodone bitartrate / acetaminophen admixture according toUSP 25
Application 4
LC_0209
PeakList: Conc.
(mg/mL)
Ret. Time
(min.)
RRT Tailing Resolution
1. unknown
7.1
2. phenol
0.15
9.8
0.88
3. morphine
0.24
11.0
1.00 1.17
3.2
Sample:
Inj.:
20.0µL
Solvent:
mobile phase
Column:
Pinnacle II
™
C18
Catalog #:
9214575-700
Dimensions: 250 x 4.6mm
Particle size: 5µm
Pore size:
110Å
Conditions:
Mobile phase: A: 0.73g 1-heptane
sulfonic acid and 10mL
of glacial acetic acid diluted
to 720mLwithwater, pH=2.33
B: methanol
(72A:28B, v/v)
Flow:
1.00mL/min
Temp.:
26.5°C
Det.:
UV@ 284nm
0
1
2
3
4 min.
LC_0211
PeakList:
Conc.
(mg/mL)
Ret. Time
(min.)
RRT Tailing Resolution Inj.%RSD (n=5)
1. acetaminophen
0.76
3.304
1.0
1.1
—
1.1
2. hydrocodone bitartrate 0.0076
6.664
2.0
1.1
17.0
0.9
Sample:
Inj.:
25µL
Sample:
500mg acetaminophenwith 5mg hydrocodone bitartrate (standard solution)
Solvent:
mobile phase
Column:
UltraC18
Catalog #:
9174575
Dimensions:
250 x 4.6mm
Particle size:
5µm
Pore size:
100Å
Conditions:
Mobile phase: A: 6.8 g potassiummonobasicwith 0.2mL of TEA per liter
B: acetonitrile
(85A:15B, v/v)
Flow:
1.5mL/min.
Temp.:
27°C
Det.:
UV@ 210nm (hydrocodone bitartrate) and 295nm (acetaminophen).
210nm channel shown
LC_0212
Baseline is
stable
210nm
