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Impurity Analysis Using a New High-Sensitivity UHPLC Detector

William Hedgepeth, Kenichiro TanakaShimadzu Scientific Instruments, Columbia, Md., USA

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Introduction

The use of UHPLC continues to grow throughout the analytical industry as researchers increase their need for faster LC separations with more sensitive detection requirements.

Recently, a new high-sensitivity detector was introduced that provides improved UHPLC system performance and includes new features that improve workplace efficiency.

The new UHPLC detector features a total reflection capillary cell to reduce system dispersion and allow ultra-high speed analysis. The high sensitivity of the detector combined with its wide dynamic detection range allows simultaneous analysis of a main peak and an impurity peak with a 0.005% concentration.

A new function of the detector provides extended dynamic range (i-DReC). This function enables an extension in apparent dynamic range by shifting the spectrum wavelength when the linearity of the calibration curve cannot be obtained due to intensity saturation of the detector.

Potential analyst error is also avoided in the dilution procedure. Results presented will demonstrate the increased sensitivity of the new PDA detector. An example of the detector’s extended dynamic range will also be presented.

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Needs for High-Sensitivity Detection

Pharmaceutical• Growing interest in impurity analysis in genotoxicity test• Publication of the related guidance by FDA ⇒ Requires analysis of trace impurities (equivalent to 0.005%)

Chemicals and Cosmetics• Increasing demand for a PDA detector for the quantitation and qualitative analysis of quasi- drugs• Simultaneous confirmation and qualitative test by retention time and spectrum

Food Inspection Agency• Necessity for analyses of impurities, toxic substances, and foreign substances with increasing awareness of food safety• Increasing analysis of harmful components such as pesticides and melamine in food due to globalization of food distribution

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High-Sensitivity Cell

Figure 1: 85 mm High-Sensitivity Flow Cell

Standard cell: 10 mm optical lengthHigh-sensitivity cell: 85 mm optical lengthThe Cell ID information can be shown with analytical results in the report.

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Sensitivity Comparison

Enhanced signal by optional high-sensitivity cell⇒ 85 mm optical length increases signal significantly

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Analysis of Ketoprofen

System: Nexera SR (CBM-20A, DGU-20A5, LC-30AD x 2, SIL-30AC, CTO-30A, SPD-M30A)

Column: Shim-pack XR-ODSIII 150x2.0mm

Mobile phase: A: 0.1% Formic acid in water, B: Acetonitrile B.Conc 30%-70% (10min)

Flow rate: 0.5mL/min Mixer: 180uL II

Column temp.: 40°C Injection volume: 2uL

Detection: 254 nm Cell: High-sensitivity cell (85mm)

Sample: Ketoprofen

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Analysis of Ketoprofen

4.50 4.75 5.00 5.25 5.50 5.75 min

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Data2:9122013_Ketoprofen_0.02mg$L_30%-70%(10min)m20a_002.lcd PDA Ch1 254nm,4nm Data1:9182013_Ketoprofen_0.02mg$L_30%-70%(10min)_002.lcd PDA Ch1 254nm,4nm

Figure 2: Chromatogram of Ketoprofen showing high sensitivity vs. standard cell

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Trace Level Impurity Detection

Figure 3: Peaks with areas of less than 0.0001% could be detected

Datafile Name:9162013_Ketoprofen_500mg$L_30%-70%(10min)rinse_039.lcdSample Name:KetoprofenSample ID:500mg/L

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min

-1

0

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6mAU

254nm,4nm

Keto

prof

en

Dataf ile Name:9162013_Ketoprofen_500mg$L_30%-70%(10min)rinse_039.lcdSample Name:KetoprofenSample ID:500mg/L

3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 min

0.0

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mAU254nm,4nm

/0.0

1980

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0013

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0003

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0162

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0026

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0005

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0007

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Keto

prof

en/9

9.90

0219

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0343

8

/0.0

1288

5

Data File Name: 9162013_Ketoprofen_500mg$L_30%-70% (10 min) rinse_039.lcdSample Name: KetoprofenSample ID: 500 mg/L

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Carryover Analysis

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 min

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Data3:9122013_Blank__30%-70%(10min)rinse_007.lcd PDA Ch1 254nm,4nm Data2:9132013_Ketoprofen_500mg$L_30%-70%(10min)rinse_020.lcd PDA Ch1 254nm,4nm Data1:9132013_Ketoprofen_0.01mg$L_30%-70%(10min)30min_002.lcd PDA Ch1 254nm,4nm

Figure 4: Ketoprofen chromatograms showing 0.01 ppm, 500 ppm, and post-run blank (top to bottom)

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Extended LinearityThe newly developed intelligent Dynamic Range Extension Calculator (i-DReC) function can expand the dynamic range in the high-concentration region.

It provides a powerful tool for checking synthetic compounds, stability testing, and the simultaneous qualification of principal components and trace impurities.

The calibration curve linearity may become lost due to signal saturation if the absorbance is extremely high, when analyzing high-concentration samples, for example.

The LabSolutions i-DReC function corrects the linearity in the high-concentration region by shifting the spectrum wavelengths and performing correction calculations using signals at wavelengths where linearity is achieved.

The i-DReC function can improve the linearity of the absorbance range over 4 AU to permit analysis of higher concentration samples.

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Extended Linearity

Using the high-sensitivity cell and the i-DReC function can further extend the applicable Nexera X2 sample concentration range.

The wider concentration range suits not only checking synthetic compounds and stability testing but also trace impurity analysis, such as the genotoxicity testing of drugs.

Labor to change dilution ratios of each compound is reduced and the risk of human error is avoided.

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Extended Linearity

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Summary

A new UHPLC detector was introduced that allows low-level impurity analysis down to 0.0001%.

The optional 85 mm high-sensitivity cell provides the highest sensitivity available in PDA detectors while keeping peak dispersion to a minimum.

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