Welcome, Guest -  Log In |  Register |  Shopping Cart
 

Food, Flavors, & Fragrances Article

Simplified LC/MS/MS Analysis of Fluoroquinolones

Using An Allure® PFP Propyl Column

By Rick Lake, Pharmaceutical Innovations Chemist, and Benjamin Smith, Applications Technician
  • Increase retention without ion-pairing.
  • Better selectivity than C18 or cyano phases.
  • Use desirable high-organic mobile phases for better ESI LC/MS sensitivity.

Fluoroquinolones are broad-spectrum antibiotics, used in both human and veterinary medicine. Because they are widely used, fluoroquinolones are target compounds in many analysis sectors, from research and clinical testing to environmental impact and residues in food. We have determined that an Allure® PFP Propyl column offers good retention capacity, and better selectivity than a C18 column, allowing simple method development strategies for fluoroquinolones.

Parent compound nalidixic acid is the structural basis for all quinolones, and fluoroquinolones are a fluorine-containing subset of this group (Figure 1). Chemically, fluoroquinolones exhibit amphoteric behavior: the nalidixic acid portion of the molecule has acidic functionality (carboxylic acid), while the compound as a whole also expresses a basic functionality. These characteristics, and the typical presence of polar functional groups, make chromatographic retention of the compounds difficult when using an alkyl phase and a simple (two-component) mobile phase. Polar groups reduce retention on alkyl phases, making a highly aqueous mobile phase, or ion-pairing, necessary for acceptable retention.

For non-selective, non-MS analyses, like potency assays, fluoroquinolones traditionally have been analyzed by reversed phase HPLC, on a C18 phase and in a highly aqueous mobile phase, as described in the USP monograph for ciprofloxacin.¹ When mass spectrometry is dictated, and a highly aqueous mobile phase is undesirable, ion-pairing with a volatile “MS friendly” reagent, like nonafluoropentanoic acid, has been used to increase retention. Although these mechanisms are sufficient, we sought to determine if, with a simple mobile phase, an Allure® PFP Propyl column would offer better retention, and possibly better selectivity, than a C18 phase.

Initially, we assayed the analytes on a C18 column, in an aqueous buffer and acetonitrile, to evaluate the retention and selectivity that could be achieved with a conventional stationary phase and isocratic mobile phase. As expected, retention was poor: an acceptable retention capacity value (roughly 2-5) required an aqueous concentration of 80% (Figure 2). Next, to see if we could improve retention through ionic interactions, we evaluated a cyanopropyl phase under the same conditions. This combination produced similar retention, but less selectivity (Figure 2). In contrast, an Allure® PFP Propyl column (pentafluorophenyl propyl phase), used under the same conditions, enabled us to achieve comparable retention capacities with the water content of the mobile phase reduced to 40% (Figure 2). In addition to greater retention capacity than the other phases, the Allure® PFP Propyl stationary phase has better selectivity — unlike with the C18 and cyano phases, there are no coelutions.

Another advantage to the Allure® PFP Propyl column’s high retention capacity for fluoroquinolones is in LC/MS analysis. Maximizing retention causes the analytes to elute in mobile phases having higher percentages of the organic component. This can increase desolvation efficiency in electrospray ionization (ESI), and can eliminate unwanted adduct formation or charge competition from matrix interferences that are less retained by the column. The result is a potential for increasing sensitivity, while using simple analytical conditions. A simple mobile phase gradient, starting with a highly aqueous content and moving to a highly organic content, can be employed to elute salts and low molecular weight sample matrix interferences ahead of the compounds of interest. We observed the same improved retention when we assayed our fluoroquinolone test mix through positive ESI LC/MS/MS on an Applied Biosystems/MDS SCIEX API 3200 triple quadrupole LC/MS/MS mass spectrometer equipped with a Shimadzu Prominence binary pump LC system (Figure 3).

The Allure® PFP Propyl phase will retain polar analytes much more effectively than a C18 phase. When greater retention is needed to give the desired selectivity, or when LC/MS analysis is desired or required, simplify your method — use an Allure® PFP Propyl column and a simple mobile phase rather than a C18 column and an ion-pairing technique.


Figure 1  The polarity of fluoroquinolones make them a challenge to retain on C18 phases.

Figure 2   Greater retention capabilities and better selectivity enable you to use simple two-component mobile phases with an Allure® PFP Propyl column.

  1. norfloxacin
  2. levofloxacin
  3. ciprofloxacin
  4. lomefloxacin
  5. enrofloxacin
  6. sparfloxacin

C18


Cyanopropyl


Allure® PFP Propyl


Sample:

Inj.:

5µL

Conc.:

~50µg/mL each component

Sample diluent:

mobile phase

Column:

Dimensions:

150 x 4.6 mm

Particle size:

5µm

Pore size:

60Å

Conditions:

Mobile phase:

10mM potassium phosphate monobasic (pH 2.5):acetonitrile, 40:60 (v:v) Allure® PFP Propyl
or 80:20 (v:v) C18, Cyanopropyl

Flow:

1.0mL/min.

Temp.:

ambient

Det.:

UV @ 220nm

Figure 3  Optimizing retention with the Allure® PFP Propyl gives high sensitivity and low matrix interference when analyzing fluoroquinolones by LC/MS/MS.

  1. norfloxacin
  2. ciprofloxacin
  3. levofloxacin
  4. lomefloxacin
  5. enrofloxacin
  6. sparfloxacin

LC_PH0426

Sample:

Inj.:

5µL

Conc.:

50ng/mL

Sample diluent:

mobile phase

Column:

Allure® PFP Propyl

Cat. #:

9169352

Dimensions:

50 x 2.1 mm

Particle size:

3µm

Pore size:

60Å

Conditions:

Instrument:

Shimadzu Prominence HPLC

Mobile phase:

A: 0.1% formic acid in water
B: 0.1% formic acid in acetonitrile

Time (min.)
  0.00
10.00
10.10
15.00

%B
10
90
10
10

Flow:

300µL/min.

Temp.:

30°C

Det.:

Applied Biosystems API 3200 Triple Quadrupole LC/MS/MS Mass Spectrometer

Ion mode:

positive

Temp.:

600°C

Ion source:

TurboIonSpray®, Electrospray 4000V



Compound
Precursor
Ion
Fragment
Ion
Declustering
Potential (V)
Collision
Energy (V)
1. Norfloxacin
319.9
276.0
233.1
36.00
36.00
23.00
35.00
2. Ciprofloxacin
332.1
288.2
244.9
41.00
41.00
23.00
31.00
3. Levofloxacin
362.1
318.1
261.0
31.00
31.00
25.00
41.00
4. Lomefloxacin
351.9
265.1
308.0
41.00
41.00
29.00
23.00
5. Enrofloxacin
360.2
316.1
245.3
36.00
36.00
25.00
37.00
6. Sparfloxacin
393.1
349.4
292.2
36.00
36.00
25.00
29.00

References

  1. United States Pharmacopoeia, 28th revision; National Formulary, 23rd edition.