Recently, a new high-sensitivity detector was introduced that provides improved UHPLC system performance and includes new features that improve workplace efficiency. This presentation introduces this technology and provides a description of its use for impurity analysis. For more information, go to www.ssi.shimadzu.com or follow Shimadzu on Twitter @shimadzussi. Thanks for viewing!

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Impurity Analysis Using a New High-Sensitivity UHPLC
Detector
William Hedgepeth, Kenichiro Tanaka
Shimadzu 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 length
High-sensitivity cell: 85 mm optical length
The 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
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
uV
Data2:9122013_Ketoprof en_0.02mg$L_30%-70%(10min)m20a_002.lcd PDA Ch1 254nm,4nm
Data1:9182013_Ketoprof en_0.02mg$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$L_30%-70%(10min)rinse_039.lcd
Sample Name:Ketoprofen
Sample ID:500mg/L
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min
-1
0
1
2
3
4
5
6
mAU
254nm,4nm
Ketoprofen
Datafile Name:9162013_Ketoprofen_500mg$L_30%-70%(10min)rinse_039.lcd
Sample Name:Ketoprofen
Sample 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
0.1
0.2
0.3
0.4
0.5
0.6
mAU
254nm,4nm
/0.019805
/0.000133
/0.000031
/0.001622
/0.000266
/0.000055
/0.000077
Ketoprofen/99.900219
/0.003438
/0.012885
Data File Name: 9162013_Ketoprofen_500mg$L_30%-70% (10 min) rinse_039.lcd
Sample Name: Ketoprofen
Sample 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
-500
0
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4500
uV
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 Linearity
The 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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