Pharmaceutical Considerations for HPLC/UHPLC Analysis

Making Adjustments to Compendial Procedures What You Can and Cannot Do Method Validation Following Multiple Adjustments to the Compendial Monograph for Atenolol and Related Impurities An Improved HPLC Method for the Determination of Ranitidine Suitable for All Dosage Forms

Reduced Solvent Consumption and Improved Productivity for USP Propranolol Hydrochloride using Core-shell Particle Columns
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Pharmaceutical Considerations for HPLC Analysis

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Introduction

With capital and operating budgets decreasing and the pressure to shorten commercialization timelines of new drugs increasing, the pharmaceutical industry faces significant challenges. Analytical chemists are asked to do more, but faster and with fewer resources. The articles, applications, products, and web resources featured in this compendium are specifically selected to help pharmaceutical scientists achieve higher productivity, better quality data, and lower operating costs to meet the current industry challenges. Selected articles include discussion of USP and EP allowable adjustments for validated methods as well as method development suggestions to achieve faster run times, decreased solvent consumption, and overall increased productivity. (pp. 3-14) Featured application notes provide total solutions ranging from sample preparation to LC/MS analysis for small molecules and biomolecules. (pp. 15-16) The recommended UHPLC/HPLC and sample preparation products are widely used by the pharmaceutical industry, to generate accurate and confident chromatographic results. (p. 17) Web resources provide thousands of searchable applications in addition to tools to select the most appropriate HPLC/UHPLC column and to optimize sample preparation protocols. (p. 18)

About the Authors

Sky Countryman has been with Phenomenex for ten years and is currently the business development manager of the PhenoLogix analytical services group. The groups responsibility is to solve analytical challenges using various chromatographic techniques. Sky has lectured all over the world on topics such as environmental testing, food safety, and pharmaceutical analysis. He is also the author of other numerous technical publications, training programs, and journal articles. Dr Philip J Koerner is the Senior Technical Manager with Phenomenex, Inc. In this capacity Dr Koerner leads a group that provides problem solving, troubleshooting, and applications support to customers around the world. In addition he has developed and presented numerous technical seminars on HPLC, GC, and SPE worldwide. Prior to joining Phenomenex in 1999, Dr Koerner spent eleven years as a research chemist with DuPont, where he was involved in HPLC and GC method development in support of Product Development and Manufacturing. Dr Koerner received a B.S. in Chemistry at the University of California, Irvine, and earned his Ph.D. in Chemistry at the University of Illinois. He is a member of the editorial advisory board for Chromatography Techniques.

About Phenomenex
Phenomenex is a global technology leader committed to developing novel analytical chemistry solutions that solve the separation and purification challenges of researchers in industrial, government and academic laboratories. Phenomenexs core technologies include products for liquid chromatography, gas chromatography, sample preparation, bulk purification chromatographic media, and chromatography accessories and equipment. For more information, visit www.phenomenex.com.

Introduction

Pharmaceutical Considerations for HPLC Analysis

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Making Adjustments to Compendial Procedures What You Can and Cannot Do

Sky Countryman and Phil Koerner Phenomenex, Torrance California, USA.

The USP and EP provide allowable adjustments that can be made to HPLC methods if system suitability requirements cannot be met. If the adjustments that are made are within the specified limits, no revalidation is necessary. The purpose of this article is to help users understand what adjustments are allowed and how to select the appropriate variable to adjust when problems are encountered.
Each of the Pharmacopeias publishes a book of standards for generic pharmaceuticals. The specifications that must be met are outlined in the monograph for each product and an analytical procedure is given to perform the assay. When chromatography is used to assay the product, the monograph also specifies certain system suitability requirements that must be demonstrated before any testing can be performed. In some cases
3

a laboratory may have problems meeting these requirements and will have to make adjustments to their method in order to meet system suitability. USP and EP have provided specific guidelines as to the allowable adjustments that can be made to a chromatographic system before revalidation is necessary (Table 1). As revalidation can be a very costly and time-consuming process, it is important to stay within these guidelines when making

adjustments to the chromatographic system. An important distinction to note is that the allowable adjustments can only be made in order to meet system suitability requirements, not to simply help speed the analysis or reduce solvent costs (although these may result). To help better understand how these adjustments can be applied to problematic methods, lets look at the example of the EP Monograph for Glibenclamide

[1] (Glyburide), which is used for the treatment of type II diabetes. System suitability requirements for the assay require that resolution between Glibenclamide and Gliclazide must not less than 5. The first column chosen for the assay was a 5 m BDS C18 100 x 4.6, but it was not able to provide the resolution necessary to meet the system suitability requirements (Figure 1). In an HPLC separation, resolution is determined by three
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Phenomenex Compendium

Table 1

Parameter Mobile Phase pH Concentration of Buffer Salts Ratio of Components in Mobile Phase

EP Allowable Adjustment 0.2 units 10% 30% of the minor component(s), or 2% absolute of that component, whichever is greater, but a change in any component cannot exceed 10% absolute. Increased to as much as twice the volume specified, provided no adverse effects; must be within stated linearity range of the method. 10 C 70% 25% 50% 50%

USP Allowable Adjustment 0.2 units 10% 30% of the minor component(s), but a change in any component cannot exceed 10% absolute (in relation to the total mobile phase content). Can be reduced as far as is consistent with accepted precision and detection limits. 10 C 70% 25% 50% 50%

Injection Volume

Column Temperature Column Length Column i.d. Particle Size Flow Rate Table: 1 Allowable changes to a chromatographic system.
Figure 1

4
RS = 4.47

1 2

Time (min)

10

Figure 1: HPLC assay for Glibenclamide on a 5 m 100 x 4.6 mm C18 column (Hypersil BDS), flow rate: 0.8 mL/min. Peaks: 1 = Glibenclamide impurity A, 2 = Glibenclamide impurity B, 3 = Glibenclamide, 4 = Gliclazide.

200

main parameters: efficiency, selectivity, and retention time (Equation 1). As the selectivity of the column is specified as C18, it was only possible to change column efficiency or retention time in order to meet 4 the system suitability requirements. 3
4

However, increasing the analysis time is typically not a desirable outcome because of decreased productivity and increased solvent consumption. For this reason increasing column efficiency was chosen as the best option.

Column efficiency can be increased using a longer column (more separation power) or smaller particle size (Equation 2). Because increasing column length would also likely increase retention time and potentially cause the previously mentioned problems, a reduction in particle size was selected as the best option to meet system suitability requirements. The EP allows for up to a 50% reduction in particle size, which allowed for a reduction in particle size to 2.5 m. Therefore, the separation of Glibenclamide was evaluated on a 2.6 m 100 x 4.6 mm C18 column where all other chromatographic parameters specified in the monograph remain unchanged (Figure 2). The particle in this column was of the partially porous (core-shell) type available from

several different manufacturers. In contrast to fully porous particles, these particles have a solid core surrounded by a porous shell, designed to reduce the diffusion path, resulting in a greater reduction in plate height than is expected from the decrease in particle size alone. Using this 2.6 m column, resolution between Glibenclamide and Gliclazide was increased to 6.89, which significantly exceeded the system suitability requirement. Back pressure increased slightly as was to be expected because of the smaller particle size, but remained well below the system limitation. All adjustments were within the Phamacopeial guidelines and the resulting method met the requirements of the monograph. The USP updates its monographs from time to time through a process of public review and comment. Proposed revisions are first published in the Pharmaceutical Forum (PF), where the stakeholders (pharmaceutical manufacturers) can review and provide comments on the proposed revisions. The feedback is then summarized and reviewed by the USP expert committee and used to help shape the final monograph. USP General Chapter <621> lists the allowable adjustments for chromatographic methods and is currently being reviewed for possible changes in PF, 35(6) (Nov.-Dec. 2009) [2]. The discussion is being fueled by the Stimuli To The Revision Process titled, Transfer of HPLC Procedures
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mAU

Phenomenex Compendium

Equation 1

Table 2

k N -1 x x R= 4 k+1
Equation 1: Where N is efficiency, k is capacity factor (retention time), and is selecitivity.
3
Equation 2

Time (min)
mAU

%Mobile Phase A 45
1 2

0-15 15-30 30-40 0

0

200

%Mobile Phase B 55 95

5 5
4 Time (min) 6

95

10

4
RS = 4.47

Table 2: HPLC parameters for Glibenclamide assay. Injection volume: 10 L; column temperature: 35 C; flow rate: 0.8 mL/min; UV detection: 230 nm; Gradient: A = mix 20 mL of a 101.8 g/L solution of freshly distilled triethylamine adjusted to pH 3.0 using phosphoric acid and 50 mL acetonitrile, dilute to 1000 mL with water; B = 20:65:15 mobile phase A/water/acetonitrile.
Figure 3

mAU

Time (min)

mAU 0 20 40 60 80 100120

N = L/(dp * h)
1 2 2 4 6

10

4
RS = 6.0

Equation 2: Where L is the length of the column, dp is the particle diameter, and h is reduced plate height.
Figure 2

0.0

1.0

2.0

3.0 4.0 Time (min)

5.0

6.0

7.0

200

Figure 3: HPLC assay for Glibenclamide on a 2.6 m 50 x 4.6 mm C18 column (Kinetex), flow rate: 1.2 mL/min. Peaks: 1 = Glibenclamide impurity A, 2 = Glibenclamide impurity B, 3 = Glibenclamide, 4 = Gliclazide.

Time (min)

10

Figure 2: HPLC assay for Glibenclamide on a 2.6 m 100 x 4.6 mm C18 column (Kinetex), flow rate: 0.8 mL/min. Peaks: 1 = Glibenclamide impurity A, 2 = Glibenclamide impurity B, 3 = Glibenclamide, 4 = Gliclazide. 5 Making Adjustments to Compendial Procedures
R = 6.0

to Suitable Columns of Reduced Dimensions and Particle Sizes [3], which discusses the benefits of using smaller particle sizes to achieve faster analysis and reduce solvent consumption. While debate over the concepts discussed in the Stimuli are interesting and should be continued, they are far from being adopted. However, there are additional adjustments to the chromatographic

system that were allowable by the current EP and USP guidelines. To determine the effect of these adjustments on the Glibenclamide assay, a 2.6 m 50 x 4.6 mm coreshell C18 column was used; the shortest column offered within the 70% limitation on column length. The flow rate was also increased by the maximum allowable amount ( 50%) to 1.2 mL/min. This resulted in a total of three adjustments from
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mAU

0120

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the original monograph (particle size, column length and flow rate). Care should be taken when making multiple changat once as they could cause unforeseen adjustments in method performance. In this case, making multiple adjustments did not seem to cause any problems with the resulting assay. Resolution between Glibenclamide and Gliclazide was reduced slightly to 6.0, but still exceeded the system suitability requirements. The resulting separation time was reduced by 57% to only 1.89 minutes, increasing sample throughput more than twofold. Solvent consumption was also reduced by 35%. Table 3 summarized the results of each of the adjustments made in these experiments and their effect on column efficiency, resolution and run time. Conclusion If problems are encountered with a chromatographic procedure that prevent you from meeting the system suitability requirements of a monograph, there are adjustments you can make that will not require revalidation. When making adjustments, consider the reason for not meeting system suitability and make strategic choices to improve method performance. Care should be taken when making more than one adjustment at a time because of the potential impact on method performance. If you make multiple
6

Table 3

Column 1 Column Dimensions Particle Size Flow Rate Backpressure Rs of Glibenclamide and Gliclazide adjustments to the method, it is probably wise to evaluate critical performance criteria such as linearity, range, precision and accuracy before using the method to test your product. In extreme cases, this might require the method to be revalidated. References [1] European Pharmacopeia 6, Glibenclamide Monograph 01/2008:0718 corrected 6.0. [2] Pharmaceutical Forum, Vol. 35(6) [Nov.-Dec. 2009], United States Pharmacopeia. [3] U.D. Neue, D. McCabe, V. Ramesh, H. Pappa and J. DeMuth, Pharmaceutical Forum, Vol. 35(6) [Nov.-Dec. 2009], 1622-1626. Note: If you have ideas or suggestions regarding the proposed revisions to USP General Chapter <621>, make sure your voice is heard. All correspondence regarding this Chapter should be addressed to Dr 100 x 4.6 mm C18 5 m fully porous 0.8 mL/min 36 bar 4.47

Column 2 100 x 4.6 mm C18 2.6 m partially porous, core-shell 0.8 mL/min 124 bar 6.89

Column 3 50 x 4.6 mm C18 2.6 m partially porous, core-shell 1.2 mL/min 64 bar 6.0

Table 3: Summary of method adjustments (for more details see text).

Horacio Pappa, Senior Scientist, US Pharmacopeial Convention, 12601 Twinbrook Parkway, Rockville, Maryland, 20852-1790, USA. Tel. +1301-816-8319 Email: hp@usp.org

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Method Validation Following Multiple Adjustments to the Compendial Monograph for Atenolol and Related Impurities
Sky Countryman and Phil Koerner, Phenomenex, Torrance, California, USA.

Previously we addressed the allowable adjustments that can be made to validated pharmacopeia methods in order to meet system suitability requirements, and how to select the appropriate variable to adjust when problems are encountered.[1] It is often advantageous to combine certain adjustments to significantly improve the method, such as column length, flow, and particle size. While multiple adjustments are allowed, they are not encouraged because of the cumulative impact they can have on method performance. If multiple adjustments are made, it is probably wise to demonstrate that the new method provides equivalent results to the old method. To do this, critical performance criteria described in USP General Chapter <1225> should be evaluated. In extreme cases, this might require method validation as it can be argued that making multiple adjustments has resulted in a completely new method.
Method Background User reports resolution problems from batch to batch of System Suitability Standard Variability seems to be associated with concentration of Impurity I, high concentration of impurity I caused resolution problems between impurity I & J System Suitability Resolution Requirement: NLT 1.4 (Impurities J & I) Research into the problem revealed an unknown impurity co-eluting with Impurity I As resolution was the primary concern between the impurities, there were several possible adjustments that could be made in order to achieve better resolution. We first attempted to improve resolution by changing the mobile phase composition and the temperature, but neither was successful in resolving the unknown impurity co-eluting with Impurity I. This
7

required changes in the HPLC column in order to achieve better resolution. Increasing column length would have improved separation, but would also have increased analysis time substantially so we chose instead to reduce particle size. Switching to 2.6 m core-shell particles allowed us to significantly improve the resolution between all three impurities. Resolution increased enough to allow the column length to be shortened from 150 to 100 mm. By combining this adjustment in column length with an increase in flow rate from 0.6 to 1.0 mL/min, the runtime was reduced from about 35 min to less than 12 min. The new method increased the number of samples that could be run per day from 41 to 60, a 50% increase. It should be noted that this adjustment to the flow rate actually exceeds the maximum allowable adjustment per USP and EP. The

adjustments made in particle size, column length and flow rate illustrate the benefits of multiple adjustments; however, one can now argue that the original method has been changed significantly. To ensure that the new method conditions would still provide equivalent results, the following
Figure 1

validation parameters were evaluated: accuracy, precision, specificity, detection limit, quantitation limit, linearity and range. Accuracy and precision were determined at three concentrations over the expected quantitation range of the method. Data is shown for the standard at the test concentration.

Figure 1: Original method for atenolol. Phenomenex Compendium www.phenomenex.com

Compendial Monograph for Atenolol and Related Impurities

Figure 2

Figure 3
120

R2 = 0.9996
100

80

R2 = 0.9969

Peak area

60

40

20

0 0 20 40 60 80 Percentage of Target load L1 5m,150x3.9mm Linear (Core-Shell L1 2.6m , 150x4.6mm) Core-Shell L1 2.6m , 150x4.6mm Linear (L1 5m,150x3.9mm) 100 120 140 160

Figure 2: New optimized method.

Table 1

Reference Standard at Test Conc.

Peak Area Impurity I Old New 66 66 66 66 67 67 66.333 0.5164 0.778%

Figure 3: linearity, LOQ, range

Injection 1 Injection 2 Injection 3 Injection 4 Injection 5 Injection 6 Average Standard Deviation (SD) Relative Standard Deviation (RSD)
Table 1: Accuracy and precision. 8

63 63 63 63 64 64 66.333 0.5164 0.815%

Results were very similar with precision being slightly better on the new method (Table 1). Both methods were able to provide linearity over the range of the assay, with the new method providing improved linearity with R2 of 0.9996 (Figure 3). The limit of detection and limit of quantitation for both methods were well within the requirements for the assay. Specificity for the new method was improved as demonstrated by the ability to resolve the final impurity. Conclusion When problems are encountered with a chromatographic procedure that prevents system suitability requirements from being met, the USP and EP have outlined allowable adjustments that can be made to

the method that will not require revalidation. However, multiple adjustments may need to be made and in such instances it is probably wise to evaluate critical performance criteria such as linearity, range, precision and accuracy before using the method to test your product. References
[1] S. Countryman and P. Koerner; Separation Science Pharma, Vol. 2(1), 2-6. [2] E. Abbasi, J. Layne, H. Behr, and S. Countryman; LC-GC: The Application Notebook, September 2009, 48-49.

Compendial Monograph for Atenolol and Related Impurities

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An Improved HPLC Method for the Determination of Ranitidine Suitable for All Dosage Forms
Sky Countryman and Phil Koerner, Phenomenex, Torrance, California, USA
Figure 1

Ranitidine hydrochloride is used to treat stomach disorders such as ulcers and oesophageal reflux disease. As this drug is now a generic, the USP and EP have developed monographs to support its formulation. The USP lists several different procedures for determination of ranitidine depending on the dosage form - oral solution, tablets, capsules, or injectables. The assay for ranitidine is done using HPLC, while chromatographic purity is established using TLC.
The lack of a robust protocol that can be used for both Assay and Purity with all dosage forms has been a major limitation of the current monographs. The USP has received a significant amount of feedback on the current protocol, but no suitable alternative has been provided. This work represents what we believe to be the required solution- a single HPLC procedure that is suitable for all testing requirements and dosage forms. Results and Discussion The current HPLC procedure in the USP monograph is similar to the EP monograph using a fully porous 3.5 m 100 x 4.6 mm column [1]. In previous articles
9

6
50

Column: fully porous 3.5m C18, 100 x 4.6 mm Conditions as per USP Monograph f or Ranitidine HCI

40

30

20

3 1 2

4 5

10

we have demonstrated improved performance for monograph methods by making adjustments that are within the allowable limits as defined in USP General Chapter <621> [2, 3]. However in this case, the changes being proposed are great enough that they require a full revalidation of the methodology. The USP Ranitidine Resolution Mixture reference standard contains ranitidine hydrochloride and four related impurities: ranitidine amino alcohol hemifumarate, ranitidine diamine hemifumarate, ranitidine N-oxide, and ranitidine complex nitroacetamide. This was prepared as indicated in the current monograph. Ranitidine S-oxide (ranitidine impurity C) was obtained from USP

-10

1. Amino alcohol hemifumarate 2. Diamine hemifumarate (Impurity A) 3. S-oxide (Impurity C) 4. N-oxide 5. Complex nitroacetamide 6. Ranitidine HCl

50

40

30

Column: core-shell 2.6 m C18, 100 x 4.6 mm Flow rate: 1.5 mL/min, UV: 230 nm, Temp: 35 C Mobile phase: A = 40 mM Potassium phosphate (pH 7.1):Acetonitrile (98:2) B= 40 mM Potassium phosphate (pH 7.1):Acetonitrile (78:22) Gradient: 100% A to 100% B in 10min, hold 5 min

20

3 1 4 2 5

10

-10

Figure 1: Comparison of the current USP method (top) using the fully porous 3.5 m column (Waters Xterra) with the method (bottom) described here using the 2.6 m core-shell column (Phenomenex Kinetex). Phenomenex Compendium www.phenomenex.com

HPLC Method for the Determination of Ranitidine

Figure 2

Average Area Count vs. % of Target Load of Ranitidine HCl

4000

3500

3000

Average Area Count

2500

R2 = 1

2000

1500

1000

500

0 0 50 100 % of Target Load 150 200 250

Figure 2: Linearity (area counts versus Ranitidine load) for the analysis of Ranitidine over the range 10200% of target sample loading.

were made at the 75, 100, and 125% concentrations and the relative standard deviation (RSD) was calculated to be 0.15, 0.10, and 0.13%, respectively. These low RSD values were well within the stated requirement of the current monograph of not more than 1.0%. Method robustness was investigated by intentionally adjusting the pH of the phosphate buffer solution 0.3 units. At pH 6.8, ranitidine Impurity A (peak 2) and ranitidine S-oxide (ranitidine impurity C, peak 3) co-eluted, as did ranitidine N-oxide (peak 4) and ranitidine complex nitroacetamide
Figure 3

(peak 5). At pH 7.4, co-elution of ranitidine Impurity A and ranitidine S-oxide occurred. This highlights the importance of controlling the phosphate buffer pH for obtaining reproducible results; therefore, the working pH should be 7.1 0.1. The mobile phase gradient rate was also adjusted by 10%, which produced very small shifts in retention time, but had no impact on resolution. Method ruggedness was demonstrated by having two analysts run the ranitidine method on two different HPLC systems and different days. The results indicate that the method is stable and provides

and added to the Resolution Mixture for method development. The overall chromatographic separation of the new method is improved (Figure 1). In the previous method, peak shape for Ranitidine Impurity A was very poor, which caused problems with quantitation. The new method improved peak shape allowing for an increase in signal-to-noise from 11.6 on the fully porous column to 77 on the Kinetex core-shell column, which represented >700% increase. Resolution of different excipients found in the various dosage forms was also difficult using the old method. Because of the higher efficiency obtained using the coreshell column, peak capacity for this gradient method was increased from 136 to 211. The increased
10

peak capacity provides increased chromatographic resolution, allowing for improved separation of additional R2 = 0.9996 impurities or product degradants. The required resolution for the old method between ranitidine R2 = 0.9969 N-oxide and ranitidine complex nitroacetamide was 1.5, and this is easily achieved (resolution = 5.0) with the new method. In order to verify applicability of this new method, several important validation parameters outlined in General Chapter <1225> were performed including linearity, range, precision and accuracy. Method ruggedness and robustness were also evaluated to further demonstrate method stability. The range of the assay was set from 10-200% of the test concentration Figure 3: Method ruggedness was demonstrated by running the Ranitidine analysis on two different HPLC systems, on different days, by two analysts. (Figure 2). Six replicate injections
120 100 80
Peak area

60

40

20

20

40

60

80

100

120

140

160

Percentage of Target load

L1 5m,150x3.9mm

Core-Shell L1 2.6m , 150x4.6mm Linear (L1 5m,150x3.9mm)

Linear (Core-Shell L1 2.6m , 150x4.6mm)

HPLC Method for the Determination of Ranitidine

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Figure 4

6
50

40

30

20

Sample: 1 Amino alcohol hemif umarate 2 Diamine hemif umarate (Impurity A) 3 S-oxide (Impurity C) 4 N-oxide 5 Complex nitroacetamide 6 Ranitidine HCl

10

-10

TRADE
to the

UP

50

40

30

20

2
10

-10

Figure 4: Assay for Ranitidine in Zantac 75 mg tablet dosage form (top), 0.1N NaOH degraded tablet (bottom)

References
[1] USP33-NF28, Monograph for Ranitidine Hydrochloride. [2] S. Countryman and P. Koerner; Separation Science Pharma, Vol. 2(1), 22-27. [3] S. Countryman and P. Koerner; Separation Science Pharma, Vol. 2(7), 9-10.

2011 Phenomenex, Inc. All rights reserved. Kinetex is a trademark of Phenomenex, Inc.

reproducible results (Figure 3). Small changes in retention time are the result of differences in system configuration; however quantitative results were similar. This method was applied to the assay of ranitidine in one readily available dosage form, Zantac 75 mg tablets, with the resulting chromatogram shown in Figure 4. This method was also applied to a sample that had been degraded by refluxing in 0.1N NaOH for 24 hours. The resulting chromatograms show an increase in the diamine hemifumarate (Impurity A) and complex nitroacetamide impurities (Figure 4).

Conclusion We are suggesting that the testing protocols in the existing Ranitidine HCl monographs be replaced with this new and improved method. While we are currently evaluating this new method for different ranitidine dosage forms, we have limited access to impurities that may be unique to each companys manufacturing process.

sub-2 m core-shell UHPLC columns on the market.

FIRST and ONLY

1.7 m

Kinetex 1.7 m core-shell columns have been shown to

outperform sub-2 m fully porous columns by 20 %*.

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HPLC Method for the Determination of Ranitidine

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Reduced Solvent Consumption and Improved Productivity for USP Propranolol Hydrochloride using Core-shell Particle Columns
Deborah Jarrett, Jeff Layne, Philip J. Koerner and Sky Countryman Phenomenex, Torrence, California, USA.

Beta blockers, such as Propranolol hydrochloride (propranolol), have been used in the treatment of hypertension and are available as generic drugs from several different vendors around the world [1]. Propanolol has also been used illegally by athletes and other persons in high stress positions to calm nerves and reduce shakiness. The United States Pharmacopeia (USP) has published a monograph for the assay of propranolol hydrochloride in drug formulations [2]. In this article, we will show how the application of fast LC techniques can be used to increase sample throughput while reducing solvent consumption.

Results and Discussion As with all USP Monographs there are specific system suitability requirements that must be met before an assay can begin. The USP assay of propranolol requires resolution to be not less than 2.0 between procainamide and propranolol, and the tailing factor for propranolol must not exceed 3.0. These values are determined by injection of the Resolution Solutions (RS) containing these compounds. The assay of propranolol hydrochloride was performed per
12

the USP monograph using a fully porous 250 x 4.6 mm 5 m C8 column, and the chromatogram of the RS is shown in Figure 1. The separation is completed in 10 minutes and resolution and the peak shapes of procainamide and propranolol are well within the system suitability requirements. At 1.5 mL/min the method uses about 15mL of solvent or 2.16 L/day to run only 144 samples. The mobile phase and its disposal costs are the primary expense in this analysis and can run into the thousands of dollars before

the column must be changed. There have been significant advances in HPLC technology over the past several years that have allowed labs to reduce their analysis time and corresponding solvent usage, while maintaining chromatographic performance. One such technology is the coreshell particle, which provides the efficiency of a sub-2 m particle at a back pressure that is compatible with many standard HPLC systems. USP Chapter <621> states that particle size can be adjusted as much

as 50% if necessary to achieve system suitability requirements. A smaller particle size will yield significant increases in column efficiency and resolution. USP <621> also states that column length may be changed +/- 70% and because analysis time is directly proportional to column length in an isocratic separation, using a shorter column will dramatically reduce analysis time. As the reduction in column length will also decrease resolution, combining these two variables is a powerful way to reduce analysis time and solvent
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Figure 1

Figure 2

2 300
mAU 100

mAU 200

Time (min)

10

100

Time (min)

10

Column:

Fully porous 5 m C8, 250 x 4.6 mm Hydrochloride

Column: Mobile Phase: Flow Rate: Inj. Volume: Temperature: Detection: Sample: 1.

Core-shell 2.6 m C8, 100 x 4.6 mm as specified in USP Monograph for Propranolol Hydrochloride 1.5 mL/min 20 L 25 C UV @ 290 nm Procainamide Propranolol

Mobile Phase: as specified in USP Monograph for Propranolol Flow Rate: 2 Inj. Volume: 1.5 mL/min 20 L 25 C UV @ 290 nm Procainamide Propranolol

300

Temperature: Detection: Sample: 1.

2.

mAU 200

2.

100

Figure 1: USP Resolution Solution on a fully porous 5 m C8 250 x 4.6 mm . 13 Reduced Solvent Consumption and Improved Productivity
0

Figure 2: Resolution Solution on the core-shell 2.6 m C8 100 x 4.6 mm column (Kinetex, Phenomenex). Phenomenex Compendium www.phenomenex.com

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PA85440211_L_1

usage while maintaining adequate resolution to fulfill suitability requirements. To test this theory, the assay of propranolol was performed on a core-shell C8 2.6 m 100 x 4.6 mm column (Kinetex, Phenomenex) using the mobile phase conditions specified in the USP monograph (Figure 2). This separation illustrates very nicely the performance benefits of the core-shell particles. The efficiency for propranolol with this column, using a conventional HPLC system, was 120,350 plates/ meter, in comparison with only 64,104 plates/meter with the fully porous column. The higher column efficiency allows resolution to be maintained well above the system suitability requirements, even though the column length has been reduced by more than 40%. The reduction in column length allows the analysis to be completed in less than 3 minutes. Using this optimized method on the core-shell particle column, running the same 144 samples requires only 648 mL, compared with the 2.16 L required with the fully porous column. This new method reduces costs associated with mobile phase by 70%. A second benefit of the optimized method is that sample productivity has been enhanced, which can allow instrumentation to be freed up for other purposes. The new method can analyze 480

samples/day, which means one HPLC can now analyse almost as many samples per day as three systems running the current USP Monograph. This leaves two instruments available to work on other projects. With most companies looking for ways to reduce their capital budgets, this can have a significant impact on the bottom line of an organization. Conclusions The introduction of Fast LC techniques such as the coreshell particle columns has brought dramatic benefits to chromatographers. The increase in efficiency provided by the particle allows QC departments analysing generic drug products to maintain chromatographic performance while simultaneously reducing solvent usage and freeing up capital equipment. The changes made in this article are within the limits specified by USP Chapter <621> and would not require re-validation of the chromatographic procedure. References
1. Wikepedia, http://en.wikipedia.org/wiki/ Propranolol (accessed 10/17/10) 2. USP Monograph for Propranolol Hydrochloride, USP33-NF28, 2010

are

YOU going?
It doesnt matter. Kinetex columns put the ultra into ANY system.

Which way

Core-shell technology. Six unique selectivities. 1.7 m and 2.6 m for versatility.

Featured Application Notes

TN-1054

APPLICATIONS
Optimizing the Analysis of Sugar Alcohol Excipients in Pharmaceutical Tablet Formulations Using Rezex Ion Exclusion HPLC Columns
Michael McGinley Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

Rezex ion exclusion HPLC columns are the solution for several published USP methodologies. The Rezex RPM (Pb+2) and RCM/ RCU (Ca+2) phases will give you the selectivity needed while the short Rezex RPM 100 x 7.8 mm columns will help to increase throughput.

Introduction

Tablet formulations of most major pharmaceutical drug products contain significant amounts of inactive ingredients (excipients) in their formulations. Such excipients are often used as binders to hold a tablet together or as a filler to increase the bulk volume of a tablet (especially for highly potent active pharmaceutical ingredients). Sugar alcohols, such as mannitol, sorbitol, and xylitol, are often used as fillers because of their inert properties and sweet taste1. While inexpensive and convenient, such sugar alcohols require unique methods for analysis and quantitation by HPLC due to their high polarity and lack of a UV absorbing chromophore. For such separations, ion exclusion chromatography is often used to detect and quantitate sugar alcohols. The method uses a combination of separation modes including gel filtration, ion-exchange, and affinity to resolve minor differences between the sugar alcohols. Several different USP methods have been developed that take advantage of the unique selectivity provided by ion exclusion HPLC. In this technical note, several different separations of sugars and sugar alcohols were performed that mimic the USP methods using Rezex RPM and RCU HPLC columns2.

Results and Discussion Efforts were undertaken to perform separations on different Rezex columns mimicking the different USP methods that are used to analyze excipients. Ion exclusion chromatography offers advantages over HILIC based methods in that they are isocratic, use a simple mobile phase (water), and can resolve chemically similar sugars. Depending on the metal salt used in the column (Lead {Pb+2} or Calcium {Ca+2}), one can achieve significantly different selectivities that can be used to optimize the desired separation. An example of a sugar and sugar alcohol mixture run on the Rezex RPM 300 x 7.8 mm column is shown in Figure 1. In this application, the Rezex RPM separates 11 different sugars and sugar alcohols. Note how the column separates the isomers mannitol and sorbitol (peaks 8 and 11) with over 10 minutes of separation between the peaks. This type of separation is useful when a complex mixture of sugars is used in a formulation and one wishes to elucidate all the potential components. However, the long run time may be an issue when a large number of samples must be analyzed.
Figure 1. Separation of a Complex Mixture of Sugar Alcohols on Rezex RPMMonosaccharide

Material and Methods

Analyses were performed using a HP 1100 LC system (Agilent Technologies, Palo Alto, CA, USA) equipped with an autosampler. Analytes were detected using either a Shimadzu RID10A RI detector (Shimadzu Scientific, Columbia, MD, USA) or a Polymer Labs ELS-2100 ELSD detector (Polymer Labs, Amherst, MA, USA). HPLC columns used for analysis include Rezex RPM 100 x 7.8 mm and 300 x 7.8 mm columns (USP L34). In addition, the Rezex RCM 300 x 7.8 mm and Rezex RCU 250 x 4.0 columns (USP L19) (Phenomenex, Torrance, CA, USA) were used to provide alternate selectivity. Sugars and sugar alcohols were purchased from Sigma Chemicals (St. Louis, MO, USA) and solvents were purchased from Thermo Fisher Scientific (Fairlawn, NJ, USA). The HPLC conditions used were dependent on the columns being used. For applications using the Rezex RPM and Rezex RCM columns, a flow rate of 0.6 mL/min was used. The applications using the Rezex RCU column used a flow rate of 0.2 mL/min. An isocratic method was used with water as the mobile phase and the column temperature was maintained constant between 75 C and 85 C, depending on the application run.

Column: Rezex RPM-Monosaccharide Dimensions: 300 x 7.8 mm Part No.: 00H-0135-K0 Mobile Phase: Water Flow Rate: 0.6 mL/min Detection: RI (Ambient) Column Temperature: 75 C 7. Meso-Erythritol Sample: 1. Stachyose 8. Mannitol 2. Maltose 9. Salicin 3. Glucose 10. Xylitol 4. Xylose 5. Galactose 11. Sorbitol 6. Fructose

A mixture of sugars and sugar alcohols run on a Rezex RPM 300 x 7.8 mm column. The order of analytes is: (1) Stachyose, (2) Maltose, (3) Glucose, (4) Xylose, (5) Galactose, (6) Fructose, (7) Mesoerythritol, (8) Mannitol, (9) Salicin, (10) Xylitol, (11) Sorbitol. Note the wide separation between Mannitol (8), Xylitol (10) and Sorbitol (11).

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Analyzing Sugar Alcohol Excipients in Pharmaceutical Tablets using Ion Exclusion Sugar alcohols in excipient formulations require unique methods for analysis and quantitation by HPLC due to their high polarity and lack of a UV absorbing chromophore. Rezex ion exclusion HPLC columns are the solution for several published USP methodologies. The Rezex RPM (Pb+2) and RCM/RCU (Ca+2) phases deliver the selectivity needed while the short Rezex RPM 100 x 7.8 mm columns help to increase throughput.

TN-0044

APPLICATIONS
Improved Clean Up and Recovery of Pharmaceutical Compounds From Plasma using Strata-X Solid Phase Extraction (SPE) vs. Traditional Liquid-Liquid Extraction Methods
Deborah Jarrett Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

Liquid-liquid extraction (LLE) is perhaps the most established clean up technique used in the chromatography field. Although it has been used for years, newer techniques with improved specificity towards particular analytes have allowed analysts to improve recovery and reproducibility of their samples. This work explores the benefits of solid phase extraction (SPE) as compared to LLE in a pharmaceutical setting. It was found that SPE provides cleaner extracts, higher recoveries, and better reproducibility which can greatly improve results.

Materials and Methods

The plasma pre-treatment step was the same for SPE and LLE and was comprised of filtration through a gauze cloth. Afterwards, 500 L of diclofenac, which was dissolved in 5 % Methanol, was added to 500 L of plasma, and the solution mixture was then acidified with 600 L of 1M Phosphoric acid.

Solid Phase Extraction

Introduction

The pre-treated plasma samples were further cleaned up and concentrated using SPE.
Cartridge: Strata-X 30 mg/ 1 mL Part No.: 8B-S100-TAK Condition: 1 mL Methanol Equilibrate: 2 mL Water Load: 1.6 mL Pre-treated plasma Wash: 1 mL 5 % Methanol Dry: 1 minute under vacuum at 10 inches Hg Elute: 1 mL Methanol Dry down: Dry down @ 53 C under a stream of nitrogen for 20 minutes Reconstitute: Reconstitute in 500 L of mobile phase

Diclofenac (Figure 1) is a slightly acidic (pKa = 4.0) non-steroidal anti-inflammatory drug (NSAID) that has been used as a post operative pain reliever in adult and pediatric patients. As a pain reliever, diclofenac is purported to act via the cyclooxygenase (COX) pathway by inhibition of prostaglandins. However, it also acts as a NSAID that inhibits the lipooxygenase pathway via a process that diminishes the formation of pro-inflammatory hormones. The subsequent quantization of diclofenac from the small volumes of biological matrices, such as plasma, has been of significant concern. Therefore, this article explores two popular extraction methods, solid phase extraction (SPE) and liquid-liquid extraction (LLE), for the isolation of diclofenac from plasma, using a water matrix as the control.
Figure 1. Structure of Internal Standard Flurbiprofen and Diclofenac

Liquid-Liquid Extraction

After pre-treatment, 5 mL of Hexane:IPA (95:5) was added to the pre-treated solution, which was followed by 1 minute of vortexing, and 10 minutes of centrifugation at 2,000 rpm. Subsequently, 4 mL of the top organic layer was transferred to a clean glass centrifuge tube and then evaporated to dryness under a stream of nitrogen at 53 C for 20 minutes.

Flurbiprofen (pKa = 4.2)

Diclofenac (pKa = 4.0)

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Cleanup of Pharma Compounds from Plasma using SPE vs LLE Liquid-liquid extraction (LLE) is perhaps the most established clean up technique used in the chromatography field. Although it has been used for years, newer techniques with improved specificity towards particular analytes have allowed analysts to improve recovery and reproducibility of their samples. This work explores the benefits of solid phase extraction (SPE) as compared to LLE in a pharmaceutical setting. It was found that SPE provides cleaner extracts, higher recoveries, and better reproducibility which can greatly improve results. Chiral LC-MS-MS Method Development of Stereoisomeric Pharma Compounds Five different Lux polysaccharide-based chiral stationary phases were explored in the reversed phase elution mode using mobile phases consisting of 0.1 % formic acid in acetonitrile or methanol to demonstrate the feasibility of LC/MS/MS analysis of a variety of acidic pharmaceutical racemates.

App ID 5508

TN-1101

APPLICATIONS
A Simple Method for Analyzing Aggregates of EPO (Erythropoietin) Using a BioSep-SEC-s2000 GFC Column
Michael McGinley Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

Gel filtration chromatography is the primary method used to analyze the amount of aggregate and dimer present in a therapeutic protein sample. A rugged yet simple method using a BioSep-SEC-s2000 column is presented for analyzing EPO samples to determine difference in the amounts of aggregate present in both fresh and longterm stored EPO samples.

Introduction

App ID 19136

With recent patent expirations, erythropoietin (EPO) is rapidly becoming the most widely manufactured recombinant biosimilar protein behind insulin with several companies throughout the world currently developing their version of the recombinant protein. Methods for analyzing EPO and quantitating post-translational modifications were mostly stagnant for the last 15 years since major manufacturers were reluctant to re-validate existing methods. However, with patent expirations, several new groups are free to use advances in instrument and column technologies to develop better analytical methods. Since protein aggregation is a major concern in the manufacture of any recombinant therapeutic, many groups are currently developing new methods for quantitating dimer and aggregate for EPO and other biosimilar proteins using higher-performing gel filtration media manufactured using modern technologies.

umns. Figures 1 and 2 show GFC chromatography on the BioSepSEC-s2000 column for two different samples of EPO. Figure 1 is a chromatogram of a freshly frozen EPO sample and Figure 2 is a chromatogram of an EPO sample that had been frozen for over a year. While one can see peaks for aggregate, monomer EPO, and buffer salts for both samples, note the increase in a dimer peak for EPO for the sample frozen for more than a year. This increase in EPO dimer suggests that the formulation used was less than ideal for this sample. 19136
3 mAU 4

20

15

10

Materials and Methods

All mobile phase solvents were purchased from EMD (San Diego CA) and reagents used in buffer preparation were obtained from Sigma Chemicals (St. Louis, MO). Recombinant Human EPO was purchased from either Cell Sciences (Canton, MA) or Sigma Chemicals. BioSep-SEC-s2000 columns (300 4.6 mm dimension) were used for all GFC (gel filtration chromatography) separations (Phenomenex, Torrance, CA). All samples were analyzed on an Agilent 1100 HPLC (Palo Alto, CA) with an autosampler and variable wavelength detector set at 214 or 220 nm; data was collected using ChemStation software (Agilent). The mobile phase was 50 mM sodium phosphate pH 6.8 with 300 mM sodium chloride in water running at a flow rate of 0.35 mL/min.

0 2 6 10 14 min

Figure 1. A freshly frozen EPO sample run on a BioSep-SEC-s2000 column. Note the early eluting high molecular weight protein (assumed to be EPO aggregate), the monomer EPO peak at 9 minutes RT, and the low molecular weight peak at the void of the column (assumed to be buffer salts in the diluent). Little or no dimer appears to be present.

Results and Discussion

While ion-exchange and reversed phase chromatography are typically used for identifying many of the post-translational modifications of a protein, GFC is exclusively used for identifying the aggregation state of most recombinant proteins. GFC specifically separates proteins by size-based differences in exclusion from a porous media which is directly related to the molecular weight of a species in solution. Recombinant EPO is approximately 30 kDa molecular weight in its glycosylated form (approximately 18 kDa for proteins), and any dimer of EPO would be expected to be around 60 kDa in size. A BioSep-SEC-s2000 series column was used for all separations, as it provides the largest separation window for proteins below 100 kDa molecular weight. This is in contrary to other methods in the past which often have used 3000-series GFC col-

Column: BioSep-SEC-s2000 Dimensions: 300 x 4.6 mm Part No.: 00H-2145-E0 Mobile Phase: 50 mM Sodium phosphate, 300 mM Sodium chloride, pH 6.8 Flow Rate: 0.35 mL/min Detection: UV @ 220 nm Sample: 1. HMW impurity (aggregate) 2. EPO dimer (not present in Figure 1) 3. EPO monomer 4. LMW impurity

Also note that the chromatography for the EPO sample run on the BioSep-SEC-s2000 shows good resolution between the monomer and dimer peaks of the EPO sample (in Figure 2) despite the sample being heavily overloaded to visualize the dimer peak. The assigned aggregate peak in both chromatograms is also very low level, but is well recovered and somewhat included into the pores

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Aggregate Analysis of EPO by Gel Filtration Chromatography Gel filtration chromatography is the primary method used to analyze the amount of aggregate and dimer present in a therapeutic protein sample. A rugged yet simple method using a BioSep-SEC-s2000 column is presented for analyzing EPO samples to determine difference in the amounts of aggregate present in both fresh and longterm stored EPO samples.

TN-1079

APPLICATIONS
Method Development for Reversed Phase Chiral LC/MS/MS Analysis of Stereoisomeric Pharmaceutical Compounds with Polysaccharide-based Stationary Phases
Philip J. Koerner, Kari Carlson, Liming Peng, Swapna Jayapalan, and Tivadar Farkas Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

Five different Lux polysaccharide-based chiral stationary phases were explored in the reversed phase elution mode using mobile phases consisting of 0.1 % formic acid in acetonitrile or methanol to demonstrate the feasibility of LC/MS/MS analysis of a variety of acidic pharmaceutical racemates.

Results and Discussion

Introduction

Developing simple and straightforward reversed phase chiral LC separations coupled with highly sensitive MS detection is a challenging requirement for conducting drug metabolism and pharmacokinetic studies of stereoisomers. Polysaccharide derivatives are the most widely used chiral stationary phases (CSP) due to their wide chiral recognition and high loading capacity. As normal phase is favorable for their principal mechanism of chiral recognition hydrogen bonding interaction the majority of chiral separations with polysaccharide phases are performed in normal phase using hexane and alcohol modifiers as mobile phase components. However, these mobile phases are highly flammable and are not compatible with atmospheric pressure ionization (API) MS ion sources. The current research was extended to investigate the effectiveness of an acidic mobile phase for the separation and detection of acidic stereoisomers by ESI or APCI LC/MS/MS and for method development of these applications.

Chiral LC/MS/MS Experiments Five different polysaccharide-based chiral stationary phases (Lux Cellulose-1, Lux Cellulose-2, Lux Cellulose-3, Lux Cellulose-4, and Lux Amylose-2 (Figure 1) were explored in the reversed phase elution mode for the separation of a variety of acidic compounds of pharmaceutical interest in mobile phases consisting of 0.1% formic acid in acetonitrile or methanol and MS/MS detection.
Figure 1. Structures of Polysaccharide-based Chiral Stationary (CSPs) Phases

Lux Cellulose-2 or -4 Cellulose tris (3-chloro-4-methylphenylcarbamate) or (4-chloro-3-methylphenylcarbamate)

Cl OCONH O Cl CH3 HNOCO O OCONH Cl CH3 CH3

Experimental

Lux Cellulose-1 Cellulose tris (3,5-dimethylphenylcarbamate)

CH3
OCONH O

Analytes: 15 acidic compounds of pharmaceutical interest were analyzed. The structures are shown in Figure 2 and the MS ionization and MRM transitions monitored are listed in Table 1.
Columns: Lux 3 m Cellulose-1, 150 x 2.0 mm Lux 3 m Cellulose-2, 150 x 2.0 mm Lux 3 m Amylose -2, 150 x 2.0 mm Lux 5 m Cellulose-4, 250 x 4.6 mm Lux 5 m Cellulose-3, 250 x 4.6 mm Kinetex 2.6 m C18, 50 x 2.1 mm (used for achiral analysis) Flow Rate: 0.2 mL/min (3 m, 150 x 2.0 mm) or 1.0 mL/min (5 m, 250 x 4.6 mm) flow split to 0.25 mL/min into MS/MS Temperature: 25 C Injection Volume: 5 L (150 x 2.0 mm) or 20 L (250 x 4.6 mm) Mobile Phase: 1. 0.1 % Formic acid in Acetonitrile or Methanol 2. 5 mM Ammonium bicarbonate in Acetonitrile or Methanol (achiral analysis) 3. 5 mM Ammonium formate in Acetonitrile or Methanol (achiral analysis) 4. 5 mM Ammonium acetate in Acetonitrile or Methanol (achiral analysis) Instrument: HPLC System: Agilent 1200 series equipped with binary pump and autosampler (Agilent, Palo Alto, CA) MS Detector: AB SCIEX 4000 LC/MS/MS Turbo V source with ESI or APCI probe TurbolonSpray ESI or APCI in Positive or Negative Ion Mode; MRM

CH3
O

CH3
HNOCO

CH3

OCONH

CH3

CH3

Lux Amylose-2 Amylose tris (5-chloro-2-methylphenylcarbamate)

Cl

OCONH
Cl

CH 3

HNO C O

O OCONH
CH 3

Cl

CH 3

Cl

Lux Cellulose-3 OCONH MeCH3 Cellulose tris-(4-methylbenzoate)

OO Cl Me HNOCO O O O C O O Me C O OCONH O O Me C Me Cl MeCH3

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15

Featured Applications

Phenomenex Compendium

www.phenomenex.com

TN-1091

APPLICATIONS
Improved Throughput, Productivity, and Performance for the USP Assay for Norethindrone and Mestranol using Kinetex 2.6 m C8 Core-Shell Columns
Philip J. Koerner, Zeshan Aqeel, and Jeff Layne Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

The ultra-high efficiency provided by Kinetex 2.6 m C8 core-shell columns is utilized to reduce analysis times and increase productivity for the USP assay for norethindrone and mestranol. Several alternatives to shorter column lengths and adjustments to flow rate were used to illustrate how the dramatic increase in efficiency can be leveraged to provide a balance between increasing productivity and reducing solvent usage.

Chromatographic Method: 10 L of sample was injected with isocratic chromatographic separation using 50:50 acetonitrile/water as the mobile phase at a flow rate of 1.0 mL/min. The column was maintained at 25 C with UV detection at 200 nm.
Conditions: Column: Kinetex 2.6 m C8 100 Fully Porous 3 m C8 Dimensions: Kinetex: 150 x 4.6 mm, 100 x 4.6 mm, and 75 x 4.6 mm Fully Porous: 150 x 4.6 mm Part No.: 00F-4497-E0, 00D-4497-E0, and 00C-4497-E0 Mobile Phase: Acetonitrile/Water (50:50) Flow Rate: 1.0 mL/min or 1.5 mL/min Inj. Volume: 10 L Temperature: 25 C Detection: UV @ 200 nm Instrument: Agilent 1100 Sample: 1. Norethindrone 2. Progesterone (IS) 3. Mestranol

Introduction

The introduction of Kinetex core-shell columns has brought dramatic benefits to chromatographers. The ability to obtain ultrahigh chromatographic separations on conventional HPLC systems with significant reductions in sample analysis time has been especially beneficial for laboratories tasked with the routine analysis of drug products. These laboratories typically have limited resources and can benefit greatly from faster separations and higher sample throughput. Mestranol and norethindrone tablets contain a combination of female hormones that prevent ovulation and are effective as oral contraceptives to prevent pregnancy. These products combine an estrogen (mestranol) and progestin (norethindrone) that are similar to the natural sex hormones (estrogen and progesterone) produced in a womans body. In general, a combination of estrogen and progestin has been determined to work better than a singleingredient product.

Results and Discussion

Reagents and Chemicals

All reagents and solvents were HPLC or analytical grade. HPLC grade acetonitrile and water were purchased from Honeywell, Burdick & Jackson (Muskegon, MI). USP norethindrone reference standard (RS), mestranol RS, and progesterone were purchased from US Pharmacopeia (Rockville, MD).

The ultra-high efficiency Kinetex columns are used for the USP assay of norethindrone and mestranol to highlight the improvements in throughput and productivity achievable using core-shell technology columns. Analyzing the standard solution containing norethindrone, progesterone, and mestranol using the HPLC column and conditions specified in the USP monograph for assay of norethindrone and mestranol tablets yields the expected chromatogram (Figure 1). The system suitability requirements for this assay specify that the efficiency determined for mestranol (peak 3) must not be less than 6,000 theoretical plates, and resolution between progesterone and mestranol must not be less than 5.0. The chromatographic result shown in Figure 1 demonstrates that these requirements are easily met using the fully porous 3 m 150 x 4.6 mm C8 column, with efficiency of 13,256 plates/column and resolution of 12.29, and analysis time just under 25 minutes. Figure 2 shows the separation of the standard solution on the Kinetex 2.6 m C8 150 x 4.6 mm column under the same mobile phase conditions and flow rate. The high efficiency expressed by the Kinetex core-shell particle results in significantly narrower peaks. Efficiency measured for mestranol is 27,397, and resolution between progesterone and mestranol is 18.03, easily meeting the system suitability requirements. The peak heights are also significantly greater than on the fully porous 3 m column. Overall, the separation is accomplished in less than half the time. Using a smaller particle (as much as 50 % smaller) can provide the benefits of increased efficiency and resolution, but comes with a price of significant increases in back pressure that may not be palatable to some chromatographers. However, the dramatic increase in chromatographic efficiency provided by the core-shell particle technology can be leveraged further by using a shorter length column, as much as 70 % shorter per USP General Chapter <621>. Since pressure is directly proportional to column length, using a shorter column can significantly offset increases in pressure attributable to the smaller core-shell particle diameter.

Equipment and Materials

Columns Used: A fully porous 3 m C8 150 x 4.6 mm column (as specified by the monograph) was compared with Kinetex 2.6 m C8 150 x 4.6 mm, 100 x 4.6 mm, and 75 x 4.6 mm columns. Instrumentation: Agilent 1100 Series HPLC (Agilent Technologies Inc., Santa Clara, CA), equipped with quaternary gradient pump, autosampler, column oven, and variable wavelength detector. Mobile Phase Preparation: A 50:50 mixture of acetonitrile and water is filtered and degassed. Standard Solution Preparation: A solution containing 0.055 mg/L of mestranol, 0.055 mg/L of norethindrone, and 0.28 mg/mL of progesterone was prepared by diluting accurately weighed quantities of USP Mestranol RS, USP Norethindrone RS, and progesterone in acetonitrile.

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Improved Throughput USP Assay for Norethindrone and Mestranol The ultra-high efficiency provided by Kinetex 2.6 m C8 coreshell columns is utilized to reduce analysis times and increase productivity for the USP assay for norethindrone and mestranol. Several alternatives to shorter column lengths and adjustments to flow rate were used to illustrate how the dramatic increase in efficiency can be leveraged to provide a balance between increasing productivity and reducing solvent usage. Analyzing Flouroquinolone Antibiotics by LC-MS This study demonstrates a method of rapid LC/MS analysis of a difficult class of basic pharmaceutical compounds, fluoroquinolone antibiotics. Using Kinetex XB-C18 good peak shape and separation of closely related compounds is achieved.

TN-1092

APPLICATIONS
Kinetex 2.6 m XB-C18 Core-Shell Column for the Rapid, Ultra-High Efficiency USP Assay for Ibuprofen
Philip J. Koerner, Deborah Jarrett, and Jeff Layne Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

This technical note describes the application of the new Kinetex 2.6 m XB-C18 column to the USP assay for ibuprofen to reduce analysis time and increase sample throughput and productivity, while improving the sensitivity of the assay for the determination of ibuprofen related compound C, a known degradation product with adverse health effects.

Equipment and Materials

Columns Used: A fully porous 5 m C18 250 x 4.6 mm column (as specified by the monograph) was compared with Kinetex 2.6 m XB-C18 100 x 4.6 mm column. Instrumentation: Agilent 1100 Series HPLC (Agilent Technologies, Inc., Santa Clara, CA, USA), equipped with quaternary gradient pump, autosampler, column oven, and diode array detector (DAD). Mobile Phase Preparation: 4.0 g of chloroacetic acid was dissolved in 400 mL of water (1.0 % w/v) and the pH adjusted to 3.0 with ammonium hydroxide, then 600 mL of acetonitrile was added and mixed well. This mobile phase mixture was filtered and degassed prior to use.
HPLC Conditions: Column: Kinetex 2.6 m XB-C18 100 Dimensions: 100 x 4.6 mm and 75 x 4.6 mm Part No.: 00D-4496-E0 and 00C-4496-E0 Mobile Phase: Acetonitrile/Water (600:400) with 4 g Chloroacetic acid, pH 3.0 Flow Rate: 2.0 mL/min Inj. Volume: 5 L Temperature: 30 C Detection: UV @ 254 nm Sample: 1. Ibuprofen 2. Valerophenone (IS) 3. Ibuprofen Related Compound C

Introduction

The introduction of Kinetex core-shell columns has brought dramatic benefits to chromatographers. The ability to obtain ultrahigh chromatographic separations on conventional HPLC systems with significant reductions in sample analysis time has been especially beneficial for laboratories tasked with the routine analysis of drug products. These laboratories typically have limited resources and can benefit greatly from faster separations and higher sample throughput. Ibuprofen is a weakly acidic, non-steroidal anti-inflammatory drug (NSAID) marketed under a variety of different trademark brands. Ibuprofen is available over the counter (OTC) in 200 mg doses in most countries (400 mg doses in some countries), and by prescription at higher doses. It is commonly used as an analgesic and anti-inflammatory, but can also relieve arthritis pain and reduce fever (antipyretic). At low doses (< 1200 mg/day), ibuprofen has the lowest incidence of digestive adverse drug reactions; however, common adverse effects include nausea, upset stomach or ingestion, and gastrointestinal ulceration or bleeding. Ibuprofen Related Compound C (4-isobutylacetophenone) is a known degradation product of ibuprofen that causes adverse effects in the central nervous system. As a result, its presence in ibuprofen tablets needs to be monitored and controlled over the shelf life of the product. The USP monograph for ibuprofen assay specifically requires the amount of ibuprofen related compound C in ibuprofen tablets to be limited to 0.1 % per tablet1. The ability to detect a low level concentration of such an impurity is critically impacted by the efficiency of the chromatographic column and we show here how to easily accomplish this with a Kinetex XBC18 column based on the ultra-high efficiency core-shell particle technology.

Standard Solution Preparation: A solution containing about 12 mg/mL of ibuprofen, 0.35 mg/mL of valerophenone (IS), and 0.012 mg/mL of ibuprofen related compound C was prepared by serial dilution in mobile phase. Chromatographic Method: 1 L of sample was injected with isocratic chromatographic separation using 60:40 acetonitrile/1.0 % (w/v) chloroacetic acid in water, pH 3.0 as the mobile phase at a flow rate of 2.0 mL/min. The column was maintained at 30 C with UV detection at 254 nm.

Reagents and Chemicals

Results and Discussion

All reagents and solvents were HPLC or analytical grade. Chloroacetic acid, ammonium hydroxide, and valerophenone were purchased from Sigma-Aldrich (Milwaukee, WI). HPLC Grade acetonitrile and water were purchased from Honeywell, Burdick & Jackson (Muskegon, MI). USP Ibuprofen reference standard (RS) and Ibuprofen Related Compound C RS were purchased from US Pharmacopeia (Rockville, MD).

The USP monograph for the assay of ibuprofen specifies using a 250 x 4.6 mm column packed with 5 m media containing a C18 bonded phase and an isocratic mobile phase as outlined in the HPLC Conditions above. The system suitability for this method requires resolution between ibuprofen and the internal standard (IS, valerophenone) and between IS and ibuprofen related compound C to be not less than (NLT) 2.5, and tailing factors for each peak to be not more than (NMT) 2.5. Figure 1 shows the chromatogram

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Rapid and High Efficiency USP Assay for Ibuprofen This technical note describes an improved method for the USP assay for ibuprofen to reduce analysis time and increase sample throughput and productivity. Using Kinetex XB-C18, sensitivity of the assay for the determination of ibuprofen related compound C, a known degradation product with adverse health effects , is improved.

TN-1094

APPLICATIONS
Using Core-Shell Kinetex XB-C18 HPLC Columns as a Solution for Analyzing Fluoroquinolone Antibiotics by LC/MS
Michael McGinley, Matt Trass, and Jeff Layne Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

TN-1071

APPLICATIONS
Determination of Impurities and Related Substances for Acetylsalicylic Acid (Ph. Eur. Monograph 0309): Increased Sensitivity and Resolution, Faster Analysis, and Reduced Solvent Usage Using Kinetex 2.6 m Core-Shell LC Columns
Ellie Abbasi, Jeff Layne, Heiko Behr, and Philip J. Koerner Phenomenex, Inc., 411 Madrid Ave, Torrance, CA 90501 USA

Additional Kinetex core-shell media research and product development has culminated in the introduction of unique bonded phases, such as Kinetex XB-C18. Kinetex XB-C18 is used for developing a rapid LC/MS method for separating fluoroquinolones; when compared to other competitive phases, the Kinetex XB-C18 demonstrates good peak shape and separation of closely related compounds.

LC/MS compatible mobile phase was used for the separation (A: 0.1 % formic acid in water, B: methanol) with a rapid gradient from 15 % B to 90 % B in 5 minutes. The flow rate was 0.4 mL/min and column temperature was maintained at 40 C. Standard solutions were diluted to 50 ng/mL with 15 % B prior to injection.

Methods can be improved for ultra-high performance without the need for higher-pressure capable instrumentation Within allowable modifications for system suitability, this method was improved for better resolution, higher sensitivity, and an 80 % reduction in analysis time.

Overview of Kinetex 2.6 m Core-Shell Technology

Precision Core-Shell Manufacturing The Kinetex technology is comprised of a nearly monodispersed 1.9 m solid silica core and a 0.35 m porous silica shell. This particle design results in a very stable and homogeneous packed column bed that significantly reduces peak dispersion due to eddy diffusion (the A term of the van Deemter equation). Additionally, the short diffusion path of the 0.35 m porous silica shell allows for faster kinetics of diffusion, thereby minimizing peak dispersion due to resistance to mass transfer (the C term in the van Deemter equation) (Figure 1 & 2).
Figure 1. Uniform Particle Size Distribution
3000

Introduction

Results and Discussion Introduction

Kinetex core-shell columns were introduced in the fall of 2009 and now include five different bonded phase chemistries: XB-C18, C8, C18, PFP and HILIC. The geometry of Kinetex core-shell particles are designed to deliver ultra-high performance, approaching 300K plates per meter, at pressures amenable to operation on standard HPLC systems with 400 bar backpressure limits1. Kinetex XB-C18, with its di-isobutyl C18 ligand, offers an alternate selectivity with different retention characteristics, especially for bases at low pH, to Kinetex C18 and other traditional C18 phase columns. Fluoroquinolones are a class of antibiotics that have become very popular over the last decade due their broad spectrum effectiveness on both gram positive and gram negative bacteria. Despite toxicity issues with several members of the drug class (that have been removed from the market) the most notable active pharmaceutical ingredient (API) in the class, Ciprofloaxin (Cipro), continues to grow in popularity as a front line antibiotic for G.I. track infections. Growing popularity requires an improved high-speed LC/MS method for quantitating such drugs. Fluoroquinolones present some unique separation challenges related to their chemical structure; they are polyaromatic basic compounds with an attached fluorine atom and carboxcylic acid group resulting in some zwitterionic properties2. Separations of fluoroquinolones were performed to evaluate if the reduced silanol activity of Kinetex XB-C18 column offered a better analysis solution compared to other UHPLC columns on the market.

Fluoroquinolones present a unique separation challenge because of the unique chemistry of the compounds. They contain multiple aromatic amines, a carboxcylic acid functionality, and at least one fluorine molecule. Kinetex XB-C18 media offers a possible solution for separating fluoroquinolones because the di-isobutyl C18 ligands offer significant shielding of silanol interactions, which lead to peak tailing. This is especially important in the volatile buffers preferred for LC/MS separations in order to maximize MS sensitivity. An example separation of a fluoroquinolone mixture is shown in Figure 1. Note the high efficiency and good peak shape with minimal tailing, as well as the near-baseline resolution of every component in the mixture. The high efficiency and good selectivity that Kinetex XB-C18, 2.6 m core-shell media delivers allow for a rapid separation of this mixture in less than 5 minutes. A fully porous sub-2 m, C18 media column (ACQUITY CSH 1.7 m C18) was shown in comparison in Figure 2. Note the significant peak tailing for all components in the mixture and the lower efficiency achieved in this application. For the fluoroquinolone application the Kinetex XB-C18, 2.6 m column offered the better solution; the good peak shape in the Kinetex chromatogram suggests a more inert solution for this application. The unique geometry of the XB-C18 ligand may also offer some selectivity benefits over other C18 phases, aside from the silanol-shielding effects. While not a focus of this application, the core-shell particle also offers method portability advantages over fully porous sub-2 m media. The Kinetex 2.6 m column shown here operates at significantly lower backpressure than sub-2 m media, making it suitable for both UHPLC and standard HPLC systems.

Presently, HPLC methods for the determination of impurities and related substances of drug products specified in monographs by the various Pharmacopoeia agencies typically employ LC columns packed with fully porous 3 and 5 micron (m) spherical silica chromatographic media. Due to the performance limitations of fully porous 3 and 5 m spherical silica chromatographic media, these analytical methods commonly require long analysis times to provide the required chromatographic resolution for the impurities present. Additionally, accurate quantitation of low-level impurities in routine LC-UV applications may be challenging due to the low intensity peaks generated by these columns. In recent years, smaller fully porous LC particles (sub-2 m diameter) have been introduced that offer faster analysis times and generate higher intensity peaks for better sensitivity. Unfortunately, since the smaller particle columns generate system backpressures that require specialized ultra-high pressure capable LC instrumentation, widespread adoption of this sub-2 m HPLC column technology has been slow. Recently, a newly developed Kinetex 2.6 m core-shell chromatographic particle has been commercialized that offers the performance benefits of fully porous sub-2 m particles but at substantially lower operating pressures. To demonstrate the performance benefits of this new core-shell technology, a Kinetex 2.6 m core-shell C18 column was compared with a fully-porous 5 m C18 column referenced in European Pharmacopoeia [Ph. Eur.] Monograph 0309 for Acetylsalicylic acid and related substances on a conventional HPLC instrument with an upper pressure limit of 400 bar. First, to demonstrate equivalency, a Kinetex column of the closest available dimension to the column referenced was operated under the conditions specified in the monograph. Then, in order to illustrate the extent of the performance benefits of the Kinetex column, a shorter Kinetex column (by 60 %) was operated at a 50 % faster flow rate; with both column length and flow rate maintained within the adjustments allowed by the Ph. Eur. for meeting system suitability. The Kinetex column achieved 80 % shorter analysis time (greater than 5x productivity improvement) and significantly improved resolution and sensitivity versus the Ph. Eur. referenced fully porous 5 m column, while meeting the system suitability requirements.

Kinetex 2.6 m

2000 Particle Count

1000

ACQUITY 1.7 m Leading 3 m

0 1 2 3 Particle Diameter (m) 4 5 6

Kinetex 2.6 m Particle with 0.35 m Porous Shell

Cross-section Image of Kinetex 2.6 m Core-Shell Particle

All chemicals were purchased from Sigma Chemical (St. Louis, MO), except for USP test standards, which were purchased from USP (Rockville, MD). Solvents were obtained from EMD (San Diego, CA). Kinetex XB-C18, 2.6 m, 50 x 2.1 mm dimension column (Phenomenex, Inc., Torrance, CA) was compared against columns from a different manufacturer (ACQUITY 1.7 m CSH C18, Waters, Milford, MA) to demonstrate performance differences. All analyses were run on an Agilent 1200 HPLC system (Palo Alto, CA) with autosampler and column oven. MS detection was performed using an AB SCIEX API 4000 mass spectrometer (Foster City, CA) with an ESI+ interface operating in MRM mode.

Materials and Methods

2.6 m

0.35 m

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Impurity Analysis of Acetylsalicylic Acid Methods can be improved for ultra-high performance without the need for higher-pressure capable instrumentation Within allowable modifications for system suitability, this method was improved for better resolution, higher sensitivity, and an 80 % reduction in analysis time.

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