Many people pursue ideas of “efficiency” as an ideal for daily life; the same can be true in the HPLC laboratory. In this work, we demonstrate the efficiency, throughput, and reliability of a dual injection system for finished pharmaceutical products and in-process active pharmaceutical ingredients

1. Application of a Dual Injection
(U)HPLC for the Analysis of
Pharmaceuticals
Shimadzu Scientific Instruments, Columbia, MD

2. Introduction
Many people pursue ideas of “efficiency” as an ideal for daily life—ideas include how many tasks
we can accomplish in a short time frame and how we can minimize our impact on the
environment. The same can be true in the HPLC laboratory. The new Nexera Dual Injection
(U)HPLC system equipped with two injection ports (independent flow paths) allows two analyses
to be run simultaneously, and yet occupies a bench space no larger than a conventional HPLC.
To showcase the Dual Injection technique for pharmaceutical applications, the USP monographs
for active pharmaceutical ingredients (API) and final pharmaceutical products were performed.
Both potency and related compound analyses for active materials were completed
simultaneously, while assay analysis for two different final products were also completed in
parallel, which demonstrates the utility of the Dual Injection technique. The results for both
analyses are stored in a single datafile which simplifies the data analysis via Multi-Data
Reporting in the LabSolutions software.
This work also shows the successful method transfer to superficially porous (SPP) columns for
chromatographic analysis with isocratic elution. Simultaneous analyses decrease the overall
analysis time by 50% while remaining compliant with USP guidelines. This work demonstrates
the efficiency, throughput, and reliability of the Dual Injection system desired in high-throughput
environment.

3. Experimental
Experiments were performed using a Nexera (U)HPLC Dual Injection system consisting of
degasser DGU-405, LC-40B XR binary pump, column oven CTO-40C, photodiode array detector
SPD-M40 and UV-Vis detectors SPD-40, and autosampler SIL- 40C XR configured for Dual
Injection mode (Fig. 1). The data acquisition and analysis was completed with LabSolutions
Chromatography Data System software version 6.99. All standards and chemicals were
purchased from Sigma Aldrich and used without additional modifications.
Flow Path
1
Flow Path
2
Solvent Delivery Units Autosampler Column Ovens Detector A - PDA
Detector B - UV
Data File
Fig. 1. Schematic diagram of Dual Injection system configuration.

4. USP Monograph: Clindamycin Hydrochloride
Purity and Potency Analysis
Column
Premier C18, 4.6x250mm,
5μm (FP)
Velox C18, 4.6x150mm, 2.7μm (SPP)
Column oven and flow cell
temperature
40 ̊C
Detection wavelength 210 nm
Mobile phase 0.05M Potassium Phosphate Buffer and Acetonitrile (55:45), pH 7.5
Flow rate 1.0 mL/min 0.8 mL/min
Injection volume 5.0 µL 2.0 µL
Main peak elution time 11 min 5 min
Table 1. Analytical conditions for FP and SPP columns.

5. USP Monograph: Clindamycin Hydrochloride
Purity and Potency Analysis
Table 2. A) System suitability results obtained from repeated injections of standard solution of Clindamycin
Hydrochloride; B) Summary for related compounds analysis for Premier (FP) and Velox (SPP) columns.
Premier C18 (n=5) Velox C18 (n=5)
Flow Stream 1 Flow Stream 2 Flow Stream 1 Flow Stream 2
R.T., % RSD 0.04 0.04 0.04 0.04
Area, % RSD 0.13 0.04 0.09 0.08
Column Efficiency 21,373 18,065 31,044 31,447
Tailing Factor 0.997 0.933 1.149 1.002
Rs (C-B vs. 7-epi) 6.5 7.7
Rs (7-epiC vs. C) 7.1 8.5
Related Compounds Premier (FP), % Velox (SPP), %
Lincomycin 0.077 0.080
Clindamycin B 0.458 0.459
7-epiclindamycin 0.224 0.228
Impurity 1 0.110 0.109
Impurity 2 0.111 0.116
Impurity 3 0.078 0.087
Total % impurities: 1.06% 1.08%

6. Premier
Datafile Name:Cl_Vlx_AppNoteData_Aug 21 2020_Cl_Test_Cl_Assay_2.lcd
Sample Name:Cl_Test
Sample ID:Cl_Assay
1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min
0.0
2.5
5.0
7.5
10.0
mAU
210nm,4nm
Lincomycin
RC1
Clindamycin-B
RC2
7-epiclindamycin
RC3
Clindamycin
Velox
Fig. 2. Expanded
chromatograms illustrating peak
retention times and resolution of
clindamycin hydrochloride
related compounds for FP
(Premier) and SPP columns
(Velox).
USP Monograph: Clindamycin Hydrochloride
Purity and Potency Analysis

7. USP Monograph: Clindamycin Hydrochloride
Purity and Potency Analysis
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min
0
25000
50000
75000
100000
125000
uV
Premier (FP)
Velox (SPP)
Fig. 3. Overlay of chromatograms
for the potency analysis of
clindamycin hydrochloride
completed on FP and SPP column
showing reduced run time and
sharper peak with SPP column
under same analysis conditions.
✓ Saved 50% of time with simultaneous purity and
potency analysis
✓ Saved additional 50% of time with SPP column

8. USP Monograph: Guaifenesin Purity and Potency
Analysis
Flow Stream 1 Flow Stream 2
R.T., % RSD 0.09 0.06
Area, % RSD 0.71 0.59
Column Efficiency 18,987 16,613
Tailing Factor 0.987 0.946
Resolution 14.8 13.8
Column Premier C18, 4.6 x 250mm, 5 µm
Column oven and Flow cell Temp 40 ̊C
Detection wavelength 276 nm
Mobile Phase Water/Glacial Acetic Acid (990:10) and Acetonitrile
Gradient elution 0-32 min 20% ACN to 50% ACN
Flow rate 1.0 mL/min
Injection volume 2.0 µL
Table 3. A) Analytical conditions for both channels; B) System suitability results.

9. USP Monograph: Guaifenesin Purity and Potency
Analysis
5.0 7.5 10.0 12.5 15.0 17.5 min
0
500
1000
1500
2000
2500
3000
3500
uV
6.0 7.0 8.0 9.0 10.0 min
0
10000
20000
30000
40000
50000
uV
Flow path 1
Purity
Flow path 2
Assay
Figure 4. Expanded chromatograms for guaifenesin purity and assay analyses with flow path 1 and 2
respectively.

10. USP Monographs: Guaifenesin Tablets and
Guaifenesin Oral Solution
Flow Path 1, Tablet Flow Path 2, Solution
Column
Epic C18 MS,
4.6x250mm, 10μm
Epic Polar, 4.6 x 250 mm,
10 μm
Col. oven & Flow cell
Temp
40 ̊C
Detection wavelength 276 nm
Mobile Phase Water, methanol, glacial acetic acid (60:40:1.5)
Flow rate 2.0 mL/min 2.0 mL/min
Injection volume 5.0 µL 5.0 µL
Main peak elution time 3.55 min 2.97 min
Table 4. Analytical conditions for guaifenesin tablet (flow path 1, PDA) and oral solution analysis
(flow path 2, UV).

11. 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min
0
1000
2000
3000
4000
5000
6000
7000
8000
uV
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
uV
Flow path 1
Tablet
Flow path 2
Oral Solution
Fig. 5. Chromatograms showing assay of guaifenesin active ingredient with standard solution in black,
tablet and oral solution in red. The tablet analysis was completed using flow path 1 and oral solution
analysis was completed using flow path 2. The amount of API in each final product was within
acceptable range of 90-110% of labeled amount.
USP Monographs: Guaifenesin Tablets and
Guaifenesin Oral Solution

12. Conclusion
This presentation demonstrates that Nexera UHPLC equipped with Dual Injection System
significantly improves laboratory efficiency and workflow by doubling the throughput and
reducing the instrument footprint in the lab. The system allows cutting total analysis time in half
by simultaneously performing the required quality control tests for finished pharmaceutical
products and in-process active pharmaceutical ingredients, such as Related Compounds and
Potency/Assay tests.
Finally, a move to superficially porous columns for analyses using isocratic elution saved an
additional 50% of the run time on the Dual Injection System, leading to further gains in efficiency.
Time saved
Analysis
1
Analysis
1
Analysis
1
Analysis
2
Analysis
2
Analysis
2
Dual injection
Flow path 1
Flow path 2
Flow path 1
Analysis
1
Analysis
1
Analysis
1
Switching
Analysis
2
Analysis
2
Analysis
2
Normal system
Dual injection Other systems
Double sample throughput
Reduced lab footprint
Reduce # of data files per
sample
References: USP General Chapter 621, USP 43 – NF 38, 2020

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