C18 guard column

In preliminary test, the liquid chromatography system Shimadzu Prominence (Shimadzu, Kyoto, Japan) was equipped with two LC-20AD pumps, a DGU-20A₃ vacuum degasser, a SIL-HTC autosampler, a CTO-20AD column oven and a controller module.
In order to solve the back conversion problem of clopidogrel, an online SPE system (Symbiosys, Spark, Holland) was used. The system integrated the liquid chromatography unit with solid phase extraction unit including binary pumps (in this method, isocratic pump was used), an autosampler, an automatic cartridge exchanger unit, two high pressure dispensers and a column oven.
The chromatographic separation was achieved on an Eternity-2.5-C18-UHPLC column (50 mm×2.1 mm, 2.5 μm, Kromasil, Sweden) with a C18 guard column (4 mm×3 mm, 5 μm, Phenomenex, USA). The column oven temperature was set at 45 °C. The samples were kept at 4 °C in an autosampler. The mobile phase consisted of 0.04% formic acid, 3 mmol/L ammonium acetate in acetonitrile/water (65:35, v/v). Mobile phase at the first minute was set at 0.15 mL/min, and flow rate returned to 0.35 mL/min in the remaining 4.5 min. Besides, a peak focusing mode was used to obtain the acquire sensitivity. Peak focus mode included mobile phase and focus reagent (water). Focus reagent was set at 200 μL/min for 1 min, propelling by a high pressure dispenser detailed in Fig. 1¹¹ .

Liquid chromatography with tandem mass spectrometry (LC-MS/MS) was developed, and cefoperazone was used as an internal standard. Cefazolin was extracted using the liquid-liquid extraction technique (LLE) with acetonitrile and methyl-t-butyl ether. Chromatographic separation was carried out on LC-MS/MS using a Kinetex C18 column (50 × 2.1 mm i.d., 1.7 μm, Phenomenex Inc., USA) that was equipped with a C18 guard column (4.0 × 2.0 mm i.d., Phenomenex Inc., USA). The column temperature was maintained at 45°C. The mobile phase consisted of a mixture of A: 0.1% aqueous formic acid and B: 0.1% formic acid in acetonitrile, which was delivered at a flow rate of 0.4 mL.min^-1 in the gradient elution mode.[14 15 (link)]
Mass spectra were obtained using a Quattro Premier XE mass spectrometer (Micromass Technologies, UK) that was equipped with an electrospray ionization (ESI) source. The mass spectrometer was operated in the multiple reaction monitoring (MRM) mode. Sample introduction and ionization was electrospray ionization in the positive ion mode. The mass transition ion-pair was selected as m z^-1 455.00 to 322.93 for cefazolin and m z^-1 646.14 to 530.07 for cefoperazone. Validation of this method was performed as recommended by the USFDA guidelines.[16 ]