Application Note 259

Determination of N-Methylpyrrolidine in Cefepime

with Nonsuppressed Conductivity Detection

INTRODUCTION Cefepime and its related compounds have been

Cefepime (Figure 1A) is a semi-synthetic, fourth- characterized by HPLC with UV detection,5 whereas
generation cephalosporin with low toxicity and a broad the NMP degradation product in cefepime has been
antimicrobial activity range against Gram-negative and determined by cation-exchange chromatography with
Gram-positive bacteria.1, 2 However, this antibiotic is suppressed conductivity detection using a Reagent-Free™
thermally unstable and will rapidly degrade at temper- Ion Chromatography (RFIC™) system.6 The U.S.
atures ≥ 25 °C and slowly degrade at lower temperatures Pharmacopeia (USP) has proposed improving
(e.g., 4 °C).3 One of the degradation products is compendial methods for determining the limit of NMP
N-methylpyrrolidine (NMP, Figure 1B).3 The primary in cefepime hydrochloride7 and Cefepime for Injection8
concerns with NMP formation are loss of cefepime by eliminating column rinse and re-equilibration steps,
potency and potential toxicity to patients. Although thereby increasing sample throughput from 3–4 h to
NMP is metabolized to the N-oxide and cleared rapidly,4 60 min per sample.9, 10
the potential for side effects is still a health concern. This work describes the determination of NMP by
Therefore, the determination of NMP in cefepime is cation-exchange chromatography with nonsuppressed
critical to assess the purity of the pharmaceutical product conductivity detection.11 The results meet the USP
due to potential toxicity of NMP to patients. criteria for Organic Impurities, Procedure 1 (Limit
of N-Methylpyrrolidine) in the proposed methods for
cefepime hydrochloride9 and Cefepime for Injection.10
A) Cefepime B) N-Methylpyrrolidine

OCH3
N
H H H N
N S
CH3 CH3

S N O N
+
N
O
NH3 COO-

25290

Figure 1. Chemical structures of A) cefepime and

B) N-methylpyrrolidine.
EQUIPMENT CONDITIONS
Dionex ICS-2100 system* including: Columns: IonPac SCG1 4 × 50 mm
Single isocratic pump (Dionex P/N 061523)

Vacuum degasser IonPac SCS1 4 × 250 mm

(Dionex P/N 061521)
High pressure, 6-port injector
Eluent: 10 mM nitric acid/5% acetonitrile
Column heater enclosure Flow Rate: 1.00 mL/min
Conductivity cell detector Inj. Volume: 10 μL (full loop)
EO Eluent Organizer, including pressure regulator, Column Temp.: 30 °C
and 2 L plastic bottle Detector Temp.: 40 °C
AS Autosampler with sample tray temperature control Detection: Nonsuppressed conductivity
and 2 mL vial tray Background: ~3300 µS
Chromeleon 6.8 or greater Chromatography Data System
® Noise: Typically <20 nS/min
(CDS) software Backpressure: ~2300 psi
Helium or nitrogen, 4.5-grade (99.995%) or better, ELUENT AND STANDARDS PREPARATION
<5 ppm oxygen (Praxair)
Eluent Solution
Filter unit, 0.2 µm nylon (Nalgene 90 mm Media-Plus, Fill a 2 L glass volumetric flask to the mark with
Nalge Nunc International P/N 164-0020) or equivalent filtered and degassed deionized (DI) water. Remove
nylon filter 101.2 mL of DI water, add 1.2 mL of concentrated nitric
Vacuum pump (Gast Manufacturing Corp. acid (70%, 15.8 N), and mix thoroughly. Add acetonitrile
P/N DOA-P104-AA) or equivalent, for degassing eluents with mixing to the mark to produce 2 L of eluent.
Glass injection vials (1.5 mL) with caps (Vial Kit, Dionex Standard and Sample Solvent Solution
P/N 055427) The NMP stock, calibration standards, and cefepime
*This application can also be run using an ICS-1100, -1600, -3000, surrogate sample solutions use 2 mM nitric acid as the
or -5000 system. diluent. To prepare 2 mM nitric acid, add 500 g of filtered
CONSUMABLES and degassed DI water to a tared glass bottle, add 0.127 mL
IonPac® SCG1 column, 4 × 50 mm (Dionex P/N 061523) of concentrated nitric acid to the flask, mix well, and add
enough filtered and degassed DI water to make 1.0 kg
IonPac SCS1 column, 4 × 250 mm (Dionex P/N 061521) of solution.
IonPac Mixer (Dionex P/N 063443)
Standard NMP Solution
To prepare a 1000 µg/mL NMP solution, add 19 mL
REAGENTS AND STANDARDS
of 2 mM nitric acid solvent to a tared glass scintillation
Deionized water, 18 MΩ-cm resistance or higher,
vial, add 0.0200 g NMP solution to the vial beneath a
filtered and degassed
well-ventilated fume hood, and add enough 2 mM nitric
Nitric acid, Ultrex® II ultrapure reagent
acid solvent to prepare 20 g of solution. Store the stock
(VWR P/N JT6901-5)
solution at 4 ºC when not in use. Make all subsequent
Acetonitrile, UV (VWR P/N BJ015-4) dilutions of NMP stock solution gravimetrically with
Cefepime Hydrochloride Reference Standard 2 mM nitric acid for generating the calibration curve
(USP P/N 1097636), Lot H0G278 was used in and system precision. Store at 4 ºC.
this study.
DL-Arginine (Sigma-Aldrich P/N A4881)
N-Methylpyrrolidine, 97% (Sigma-Aldrich P/N M79204)

2 Determination of N-Methylpyrrolidine in Cefepime with Nonsuppressed Conductivity Detection

SAMPLE PREPARATION Column: IonPac SCG1, SCS1, 4 mm
Eluent: 10 mM nitric acid/5% acetonitrile
Cefepime Hydrochloride Samples Flow Rate: 1.00 mL/min
Inj. Volume: 10 µL
Prepare 5 mg/mL cefepime matrix solution based Column Temp.: 30 °C
on the cefepime assay result listed on the USP Reference Detection:
Detector Temp.:
Nonsuppressed conductivity
40 °C
Standard label (0.865 mg cefepime/mg solid in Lot Samples: A. 6 common cation standard diluted
in 2 mM nitric acid
H0G278). For this study, weigh 0.0289 g cefepime B. 9 µg/mL NMP in 2 mM nitric acid
hydrochloride into a tared, 20 mL glass scintillation vial Peaks: 1–6. Common cations
7. NMP 9 µg/mL
and dissolve in 5 g of 2 mM nitric acid to produce a
10 2 34 6
5 mg/mL cefepime solution. Mix to dissolve the solid,
then immediately withdraw 1.0 mL of the matrix solution 1
5
and place the aliquot in a 1.5 mL glass autosampler vial.
This vial then should be placed into the AS sample tray
cooled to 4–6 ºC and analyzed immediately to minimize
sample degradation. To determine the method’s accuracy µS

and precision, reweigh the scintillation vial, add an

appropriate weight of 1000 µg/mL NMP solution to
produce a working sample of 15 µg/mL NMP in 5 mg/mL A
cefepime, and immediately analyze the solution. 7

Cefepime for Injection Simulated Samples B

0
Retention time and peak area precision of NMP 0 5 10 15
in the simulated Cefepime for Injection samples were Minutes 27698

evaluated. In Cefepime for Injection, arginine is added at Figure 2. Chromatography of A) six common cation standard and
an approximate concentration of 725 mg/g of cefepime B) 9 µg/mL NMP standard dissolved in 2 mM nitric acid.
to maintain the pH of the constituted solution between
4 and 6. Prepare 5.0 g of 5 mg/mL cefepime solution in
2 mM nitric acid as described above. Based on the acid with 5% acetonitrile. The organic solvent reduces the
expected 0.827 mg arginine/mg arginine hydrochloride, hydrophobic interactions of cefepime with the stationary
add 0.0219 mg arginine hydrochloride to the cefepime phase to improve sample throughput, whereas higher
solution. Mix, immediately withdraw 1.0 mL of this acid concentrations decrease the retention of alkali
matrix solution, place the aliquot in a 1.5 mL glass and alkaline earth metals on the column. However, an
autosampler vial, and place the vial into the AS sample increase in the hydronium ions in the eluent produces a
tray cooled to 4–6 ºC. Start the sequence soon after proportional increase in the background conductivity and,
placing the vials in the autosampler. therefore, baseline noise, which reduces the sensitivity
of the method. Despite the reduction in sensitivity, the
RESULTS AND DISCUSSION method retains the ability to quantify at or below the USP
Chromatography specification for NMP in cefepime hydrochloride.
The IonPac SCS1 column is a low-capacity, silica- Figure 2A demonstrates the separation of common cations
based, weak cation-exchange column functionalized with on the IonPac SCS1 column using the eluent conditions
carboxylic acid groups.12 Typical recommended eluent described in the USP method. As shown, the retention
conditions for this column are 3 mM MSA at 1 mL/min time of these cations is significantly reduced relative
to elute six common cations in about 35 min (guard and to the standard conditions with 3 mM MSA. Figure 2B
analytical columns). In the proposed USP method, the shows the separation of NMP eluting at 10 min as a sharp
described eluent composition consists of 10 mM nitric peak with an asymmetry of 1.1 and a plate count > 15000.

Application Note 259 3

 Column: IonPac SCG1, SCS1, 4 mm Column: IonPac SCG1, SCS1, 4 mm
Eluent: 10 mM nitric acid/5% acetonitrile Eluent: 10 mM nitric acid/5% acetonitrile
Flow Rate: 1.00 mL/min Flow Rate: 1.00 mL/min
Inj. Volume: 10 µL Inj. Volume: 10 µL
Column Temp.: 30 °C Column Temp.: 30 °C
Detection: Nonsuppressed conductivity Detection: Nonsuppressed conductivity
Detector Temp.: 40 °C Detector Temp.: 40 °C
Sample: Simulated Cefepime for Injection Samples: A. 5 mg/mL Cefepime hydrochloride RS (unspiked)
B. 5 mg/mL Cefepime hydrochloride RS spiked
Peaks: 1. Arginine — with 15 µg/mL NMP
2. NMP 0.19%
3. Cefepime — A B
10 1
Peaks: 1. NMP 0.17% 0.46%
3 2. Cefepime — —
10
2

µS

µS
1

0 B

0 10 20 30 40 50 60 A
Minutes 0
27699
0 10 20 30 40 50 60
Figure 3. Determination of NMP in simulated Cefepime for Minutes 27700
Injection sample.
Figure 4. Comparison of A) unspiked and B) 15 µg/mL (0.3%)
NMP-spiked solutions of 5 mg/mL cefepime in 2 mM nitric acid.

Figure 3 shows the separation of NMP in a simulated 1.6 µg/mL, respectively, corresponding to 0.01 and
Cefepime for Injection sample, which also contains 0.032% in 5 mg/mL cefepime. The LOQ estimate was
relatively high concentrations of arginine. As shown, 10-fold lower than the 0.3% acceptance criterion cutoff
arginine and NMP are well resolved (Rs = 8.2) on the level for cefepime hydrochloride and more than 30-fold
IonPac SCS1 column using the conditions specified in the lower than the 1.0% acceptance criterion cutoff level for
USP monograph. This chromatogram also demonstrates Cefepime for Injection.
that cefepime elutes completely within 6× the NMP To determine method linearity, calibration standards
retention time limit as specified in the USP proposed were prepared at nine concentration levels in the range of
monograph, thus avoiding cefepime carryover.9, 10 5–100 µg/mL NMP in 2 mM nitric acid, corresponding
However, the total analysis time is twice as long as the to 0.1–2.0% in 5 mg/mL cefepime. A plot of peak area
IC method described in Dionex AN 199.6 versus concentration produced a correlation coefficient (r2)
Limit of Detection, Limit of Quantification, and Linearity value of 0.9996 using a linear least squares regression
The USP General Chapter on validation of fit. The relative standard deviation of the measured peak
compendial methods <1225> specifies a signal-to-noise areas based on the calibration curve was < 1.8%.
(S/N) ratio of three for the limit of detection (LOD) and Accuracy and Precision
10 for the limit of quantification (LOQ).13 Baseline noise Method accuracy was evaluated by spiking
was determined to be 18 nS by averaging the peak-to- 15 µg/mL of NMP into a 5 mg/mL cefepime sample
peak noise of seven system (no injection) blanks over two solution (Figure 4). The unspiked cefepime sample
1 min windows centered on the NMP retention time. Peak contained 8.41 µg/mL (0.17%) NMP, which was below
heights from triplicate injections of standards in 2 mM the acceptance criterion cutoff value. After correcting for
nitric acid were plotted versus NMP concentration. the amount of NMP in the cefepime sample, the average
The LOD and LOQ estimates for NMP were 0.5 and recovery for three replicates was 99 ± 1.0%.

4 Determination of N-Methylpyrrolidine in Cefepime with Nonsuppressed Conductivity Detection

 Table 1. Retention Time and Peak Area Precisions for N-Methylpyrrolidine in Cefepime Samples
Samplea N NMP Conc. Average Retention Retention Time Average Peak Peak Area RSD
Time (min) RSD Area (µS*min)
15 µg/mL NMP Standard 6 15.0 µg/mL 9.742 0.02 0.3686 0.4
USP Cefepime Hydrochloride RS b
6 0.18% 9.715 0.06 0.2033 1.1
Simulated Cefepime for Injectionc 6 0.19% 9.704 0.13 0.2248 1.6
a
- 2 mM nitric acid solvent
b
- 5 mg/mL cefepime
c
- 5 mg/mL cefepime + 3.6 mg/mL arginine

Table 1 summarizes the results of three sets of REFERENCES

repeatability experiments. System precision refers to 1. Sader, H.S.; Fritsche, T.R.; Jones, R.N. Potency
repeatability for a standard without cefepime or cefepime/ and Spectrum Trends for Cefepime Tested Against
arginine mixture present. Excellent system precisions 65746 Clinical Bacterial Isolates Collected in
for NMP retention time (0.02%) and peak area (0.4%) North American Medical Centers: Results from
were measured for an NMP concentration of 15 µg/mL. the SENTRY Antimicrobial Surveillance Program.
Precision of NMP retention time and peak area was also Diagn. Microbiol. Infect. Dis. 2005, 52 (3), 265–273.
determined for cefepime HCl and Cefepime for Injection 2. U.S. Food and Drug Administration. Maxipime™
samples. Retention time and peak area repeatabilities were (Cefepime Hydrochloride, USP) for Injection.
0.06% and 1.1%, respectively, for NMP in 5 mg/mL www.fda.gov/ohrms/dockets/dockets/06p0461/06p-
cefepime and 0.13% and 1.6% for NMP in 5 mg/mL 0461-cp00001-02-Attachment-01-vol1.pdf
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the autosampler tray at 4 ºC over the 6.1 h time interval T.W.; Gill, M.A. Stability and Antibacterial
needed to run the six replicates of each of the two Activity of Cefepime During Continuous Infusion.
cefepime-containing samples. NMP peak areas increased Antimicrob. Agents Chemother. 2003, 47 (6),
2.7% for the cefepime hydrochloride sample and 3.9% 1991–1994.
for the Cefepime for Injection simulated sample. Using 4. Forgue, S.T.; Kari, P.; Barbhaiya, R. N-Oxidation
the IC-based method described in AN 199 will result of N-Methylpyrrolidine Released in vivo from
in faster sample throughput, producing less cefepime Cefepime. Drug Metab. Dispos. 1987, 15 (6),
decomposition during analysis of a given sample. 808–815.
5. Dionex Corporation, Determination of Cefepime and
CONCLUSION
Cefepime-Related Substances Using HPLC with UV
This work presents the determination of
Detection. Application Note 205, LPN 2081, 2008,
N-methylpyrrolidine, a cefepime decomposition product,
Sunnyvale, CA.
using a silica-based, weak cation-exchange column
6. Dionex Corporation, Determination of
coupled with nonsuppressed conductivity detection.
N-Methylpyrrolidine in Cefepime Using a Reagent-
Results for LOD, LOQ, linear calibration range, spike
Free Ion Chromatography System. Application Note
recovery, retention time precision, and peak area precision
199, LPN 2005, 2008, Sunnyvale, CA.
determinations show that this instrumental configuration
7. Cefepime Hydrochloride; United States Pharmacopeia,
fulfills acceptance criteria for determining NMP in
The National Formulary: USP 33, NF 28, 2010.
cefepime hydrochloride and Cefepime for Injection
8. Cefepime for Injection; United States Pharmacopeia,
samples. Cefepime decomposition during sample analysis
The National Formulary: USP 33, NF 28, 2010.
can be decreased and eluent preparation can be simplified
9. In-Process Revision Briefing: Cefepime
by using the method described in AN 199.
Hydrochloride. Pharmacopeia Forum, 2010, 36 (1).

Application Note 259 5

 10. In-Process Revision Briefing: Cefepime for Injection.
Pharmacopeia Forum, 2010, 36 (1).
11. Dionex Corporation, Comparison of Suppressed
to Nonsuppressed Conductivity Detection for the
Determination of Common Inorganic Cations.
Application Note 157, LPN 1560, 2004,
Sunnyvale, CA.
12. Dionex Corporation, Product Manual: IonPac
SCS 1 and SCG 1 Columns.
Document No. 031948-05, 2006, Sunnyvale, CA.
13. Validation of Compendial Methods; United States
Pharmacopeia, The National Formulary: General
Chapter <1225>, USP 33, NF 28, 2010.

SUPPLIERS
Gast Manufacturing Corp., 2550 Meadowbrook Road,
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Tel: 1-800-625-4327. www.nalgenunc.com
Praxair, 39 Old Ridgebury Road, Danbury,
CT 06810-5113, U.S.A.
Tel: 877-772-9247. www.praxair.com
Sigma-Aldrich Chemical Company, P.O. Box 14508,
St. Louis, MO 63178, U.S.A.
Tel: 1-800-325-3010. www.sigma.sial.com
VWR International, 1310 Goshen Parkway,
West Chester, PA 19380, U.S.A.
Tel: 1-800-932-5000. www.vwr.com
U. S. Pharmacopeia, 12601 Twinbrook Parkway,
Rockville, MD 20852, U.S.A.
Tel: 1-800-227-8772. www.usp.org

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Ultrex is a registered trademark of J.T. Baker.

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