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Good morning to all. Now I'm trying to develop quantification method for lysine aescinate injection by HPLC. Lysine peak in not eluting. If someone knows about these method parameters, please help me.
youhana Hosny
1 m ago
it can’t be detected on uv detector
are using uv detector ?
Sunnatulla Tugizboyev
1 m ago
Primesep 100 column with UV 210 nm it is working only lysine standard.
The Problem: The Pressure vs. Efficiency Trade-off
Totally Porous Particles (The Old Standard): These are like "sponges" or "Nerf balls"—pores go all the way through. To get better separation (efficiency), you need smaller particles.
The Trap: As you shrink the particle size (e.g., from 3.5 µm to 1.8 µm) to sharpen peaks, the backpressure skyrockets (often quadruples). Standard HPLC instruments (max ~6000 PSI) cannot handle the pressure required by these sub-2 micron particles.
The Solution: Superficially Porous Particles (SPP)
Also known as Core-Shell or Porous-Shell particles, these offer a "best of both worlds" solution:
The Structure: They have a solid, non-porous core (like a glass marble) coated with a thin porous shell.
Example: A 1.7 µm solid core + a 0.5 µm porous shell = 2.7 µm total particle size.
Why It Works:
Lower Pressure: The pump "sees" a larger 2.7 µm particle, keeping backpressure low enough for standard HPLC instruments.
Higher Efficiency: The sample molecules only have to diffuse through the thin outer shell (short diffusion path). This mimics the physics of a much smaller (~1 µm) particle.
Key Takeaway
Superficially porous columns allow you to achieve UPLC-level efficiency (sharp peaks) using standard HPLC hardware (lower pressure).
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In HPLC analysis, the biggest lie a chromatogram can tell you is that "bigger peak = more amount."
We all know that different molecules interact with detectors differently. Yet, assuming a Relative Response Factor (RRF) of 1.0 for impurities is still a common shortcut that can lead to dangerous under- or over-estimation of toxic impurities.
Why RRF Determination is Non-Negotiable:
Chromophore Differences: Just because it’s an isomer doesn’t mean it absorbs UV light the same way.
Wavelength Sensitivity: An RRF valid at 254 nm might be completely wrong at 210 nm.
Regulatory Impact: ICH guidelines (Q3A/Q3B) often require correction factors if the response differs by more than 10-20% (0.8–1.2).
The "Slope Method" Gold Standard: To get a true RRF, avoid the single-point check. You need to compare the slopes of the linearity curves (Impurity vs. API) to rule out matrix effects and intercept bias.
RRF = Slope (Impurity) / Slope (API)
If you aren't correcting for response factors, are you really quantifying? Or are you just guessing?
How does your lab handle RRFs for unknown impurities? Do you default to 1.0 or run full slope-method determinations?
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Sustainability in the lab is no longer just a buzzword—in 2025, it’s a necessity. With rising solvent costs and stricter waste regulations, Green Chromatography is becoming the new gold standard in pharmaceutical R&D and QC.
3 Trends defining the Green Shift:
Solvent Swaps: We are seeing successful validations replacing traditional solvents like Acetonitrile and Methanol with greener, bio-renewable alternatives like Ethanol and Dimethyl Carbonate (DMC).
Miniaturization: The shift to micro-LC and nano-LC is cutting solvent consumption by up to 90% without sacrificing resolution.
Core-Shell Efficiency: Using sub-2 µm core-shell particles to speed up runs and drastically reduce mobile phase usage.
It’s not just about saving the planet; it’s about saving the budget.
Call to Action: What’s the biggest challenge you face in making your methods greener? Let's discuss in the comments. ?
#GreenChemistry #HPLC #Sustainability #Laboratory #PharmaRD #GreenHPLC #AnalyticalChemistry #2025Trends
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Suggest Mobile Phase for Sertraline HCl Impurity Method for, with good separation between "USP Sertaline HCl Racemic Mixture and Sertraline HCl".
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During batch file run after five std. Run pressure drop occurs due to which shift in RT. How can we file an incident report. What reason and explanation and remedies we file
youhana Hosny
2 m ago
Is there any source of leak detected ?
Babak Astani
1 m ago
✅ Incident Report – HPLC Pressure Drop & RT Shift
1) What happened (Incident Description)
During a batch sequence, after the injection of five standards, a sudden pressure drop occurred in the HPLC system. Following the pressure drop, a shift in retention time (RT) was observed for subsequent standard injections and samples. The batch was stopped, and the issue was investigated.
✅ 2) Probable Cause(s)
A) Partial blockage dislodged → sudden flow increase → pressure drop
Sometimes a partially clogged column, guard column, or inline filter gets cleared suddenly.
When the blockage clears, backpressure drops and retention times shift to lower RT because of increased actual flow.
B) Leak in the system
A loose fitting, worn ferrule, or micro-leak in pump head or column inlet can reduce pressure.
Leak causes unstable flow → RT shift.
C) Pump malfunction / air bubble
Air bubble entering pump head reduces pressure and flow.
Causes inconsistent RT.
D) Mobile phase composition shift
Sudden change in gradient accuracy due to pump mixing issue.
Leads to RT drift and pressure change.
✅ 3) Explanation (Technical Justification)
HPLC pressure is directly linked to flow resistance across the column.
A sudden pressure drop indicates reduced flow resistance, typically due to:
Clearance of blockage,
Leak formation, or Pump instability.
Retention times shifted because the actual flow rate changed even though set flow seemed unchanged.
This directly impacts chromatographic performance and validity of the batch.
✅ 4) Remedies / Corrective Actions (CA)
You can list these:
1. Checked entire fluidic path (inlet filter → pump → mixer → injector → column → detector) for leaks.
2. Tightened all fittings and replaced worn ferrules.
3. Purged pump thoroughly to remove air bubbles.
4. Flushed column with strong solvent (e.g., 100% organic) to remove particulate blockage.
5. Cleaned / replaced guard column or inline filter if excessive debris was found.
6. Verified flow rate accuracy using a volumetric flask test.
7. Re-equilibrated column and re-injected system suitability standards.
8. Ensured no further pressure fluctuations before re-running batch.
✅ 5) Preventive Actions (PA)
Regular cleaning of inline filters.
Scheduled column maintenance (backflushing if allowed).
Strict filtration of mobile phase & samples through 0.22 µm filters.
Routine pump maintenance (seal wash, piston seal inspection).
Daily leak check before starting
In analytical method development, particularly for analyzing active pharmaceutical ingredients (APIs) and their impurities, the pKa value of a compound is a fundamental parameter.
Why is pKa so vital?
The pKa value is the specific pH at which a molecule exists in a state of equal equilibrium between its ionized and unionized forms (50% ionized, 50% unionized). This balance is crucial because the ionization state directly dictates how a compound interacts with the HPLC system, influencing its retention time, selectivity, and overall peak shape.
Key Applications of pKa in Optimizing HPLC Methods
Strategic Mobile Phase pH Selection:
Choosing a mobile phase pH relative to the analyte's pKa is essential for controlling its ionization state and, consequently, its retention and resolution.
General Guideline: For stable ionization and reproducible chromatography, it is recommended to work at a pH that is at least 2 units away from the pKa value (pKa ± 2).
Achieving Superior Peak Shape:
The unionized form of a compound is typically more hydrophobic, allowing for stronger interactions with the stationary phase. This results in sharper, more symmetrical peaks.
Conversely, the ionized form is more hydrophilic and often shows poor retention, leading to undesirable broad, weak, or tailing peaks.
Improving Selectivity Between Analytes:
Compounds with different pKa values can be effectively separated by fine-tuning the mobile phase pH.
Even small adjustments in pH can significantly enhance resolution and selectivity, facilitating the separation of closely eluting peaks.
Ensuring Method Robustness:
A method operating at a mobile phase pH close to an analyte's pKa is highly sensitive to minor pH fluctuations. This can lead to inconsistent retention times and affect system suitability.
Working at a pH away from the pKa makes the method more robust and less susceptible to these small variations.
✔ In Summary: The pKa is more than just a number; it is a powerful guiding tool. By making pKa-driven decisions, analytical scientists can optimize retention, improve selectivity, prevent peak tailing, and ensure the robustness of their HPLC methods, ultimately leading to faster development and more reliable results.
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Suggest Mobile:
Composition isocrated / Gradient for
Oteseconazole:SHR8008X:SHR8008JA retention time with good separation.
Paing Aye
2 m ago
Based on the chemical properties of Oteseconazole (VT-1161)—which is a tetrazole-based antifungal agent containing pyridine and fluorinated phenyl rings—and typical separation strategies for its related impurities (SHR8008 series), here is a robust starting point for HPLC method development.
Since specific impurity codes like SHR8008X and SHR8008JA are often proprietary manufacturing intermediates or degradation products, a Gradient Method is strongly recommended over an isocratic one to ensure you capture both early-eluting (polar) and late-eluting (non-polar) impurities.
Recommended Gradient Method (Best for Impurity Profiling)
This method utilizes a C18 column with a standard acidic buffer to suppress ionization of the basic nitrogen atoms found in Oteseconazole, ensuring sharp peak shapes.
Chromatographic Conditions:
Column: C18 Column (e.g., Agilent Zorbax Eclipse Plus or Waters XBridge), 150 x 4.6 mm, 3.5 um or 5 um.
Flow Rate: 1.0 mL/min
Detection (UV): 210 nm (for detecting impurities with weak chromophores) or 260 nm (more specific to the drug).
Column Temperature: 30^\circ\text{C} - 40^\circ\text{C}
Mobile Phase Composition:
Mobile Phase A: 0.1% Orthophosphoric Acid (H_3PO_4) in Water OR 10mM Potassium Dihydrogen Phosphate (KH_2PO_4) adjusted to pH 3.0.
Mobile Phase B: Acetonitrile (ACN).
Time (min) Mobile Phase A (%) Mobile Phase B (%) Purpose
0.0 70 30 Initial hold for polar impurities
5.0 70 30 Isocratic hold
25.0 10 90 Linear ramp to elute Oteseconazole & hydrophobic impurities
30.0 10 90 Wash step
30.1 70 30 Return to initial
35.0 70 30 Re-equilibration
Alternative Isocratic Method (For Quick Assay)
If you require an isocratic method specifically (though less effective for separating complex impurities like SHR8008X/JA), try this composition.
Composition: Buffer : Acetonitrile (45 : 55 v/v)
Buffer: 0.1% Orthophosphoric acid or 10mM Ammonium Acetate (pH 4.5).
Note: If Oteseconazole elutes too quickly (near the void volume), decrease the Acetonitrile to 45% or 40%. If it takes too long, increase Acetonitrile to 60%.
Critical Considerations for Optimization
To achieve the "good separation" requested in the prompt, consider these factors if the initial run does not resolve SHR8008X from SHR8008JA:
1. pH Control is Key
Oteseconazole contains basic nitrogen atoms (tetrazole/pyridine moieties).
If peaks are tailing: Ensure your buffer pH is roughly 3.0. This keeps the basic nitrogens protonated and prevents them from interacting with unreacted silanols on the column stationary phase.
Buffer Choice: If you are using Mass Spectrometry (LC-MS), swap the Phosphoric Acid for 0.1% Formic Acid or Ammonium Formate.
2. Modifying Selectivity
If the impurities (SHR8008X and JA) co-elute with the main peak:
Change the Organic Modifier: Substitute Methanol for Acetonitrile. Methanol has different selectivity (protic solvent) compared to Acetonitrile (aprotic) and often resolves positional isomers better.
Change the Column Chemistry: If a standard C18 fails, try a Phenyl-Hexyl column. The pi-pi interactions from the phenyl column often help separate aromatic compounds like azole antifungals better than a standard alkyl chain.
3. Sample Diluent
Dissolve your sample in a mixture of Water:Acetonitrile (50:50). Avoid dissolving in 100% Acetonitrile if your starting gradient conditions are high aqueous, as this causes peak distortion (fronting).
Lots of thanks for resolve this issue.
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In UPLC during RP-HPLC Run with mobile phase as 0.1% TFA+ Water and 0.1%TFA+ ACN with 55 C column temp., the peak keeps shifting to wards left as the sequence continues.
What could be the reason.
Initially the for few runs the RT was consistent.
Any suggestions to look in to ?
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Please Provide me the Tropicamide 1% Eye Drop HPLC related substance Method of analysis and impurities limit.
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Best mobile phase for assay and their ratio mobile phase for better' resolution
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How make integration on empower for hplc
Sanjay Kolte
3 m ago
For any Empower software related questions refer their Tips guide in describes detailed information,below is linc fir same
Empower Tip: Integrating Peaks https://share.google/ewjvPgcZUKWHoFxUI
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Now I try to analysis syrup contain Vit.D3 and calcium by hplc but i have difficulty separating to get clear result as assay
Can you give any method or tips
Hashem Yehia
4 m ago
Use mobile phase of (MeOH 96%:ACN 4%), and use solvent methane 100% measure at 265 nm with C18, 15 cm column with a flow rate of 1.5 ml/min will give you a good peak for Vitamin D3
When developing an HPLC method, we often focus on column chemistry, mobile phase composition, and detection. But what happens when the compound itself is chiral?
Enantiomers — mirror-image molecules — behave identically in most achiral environments, which means they often co-elute as a single peak on standard C18 or C8 columns. In biological systems, however, their behavior can be dramatically different:
One enantiomer may provide the therapeutic effect.
The other may be inactive, or even harmful.
This is why regulatory agencies (ICH, FDA, EMA) require chiral separation and quantification during method development and validation. Enantiomeric purity is not only a regulatory requirement but also essential for patient safety and drug efficacy.
Strategies for Chiral Separation in HPLC:
Chiral Stationary Phases (CSPs) – polysaccharide, cyclodextrin, protein, or Pirkle-type columns that enable selective interactions.
Chiral Mobile Phase Additives (CMPAs) – such as cyclodextrins forming transient diastereomeric complexes.
Indirect Approach (Derivatization) – converting enantiomers into diastereomers with chiral reagents (e.g., Marfey’s, Mosher’s) for separation on achiral columns.
Key Considerations in Method Development:
Screening multiple CSPs to identify the best selectivity.
Selecting the appropriate elution mode (normal-phase, reversed-phase, polar organic).
Optimizing pH and temperature to improve resolution.
Ensuring scalability for both analytical and preparative applications.
In modern pharmaceutical analysis, chirality is more than just a separation challenge — it is a critical quality attribute that guarantees both drug safety and efficacy.
For professionals in analytical R&D and QC, developing strong expertise in chiral HPLC method development is essential. Chirality is not just chemistry; it is directly linked to patient safety.
#HPLC #Chirality #MethodDevelopment #AnalyticalChemistry #Pharma #DrugSafety #QualityControl
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Impurities are unwanted chemicals that may be present in drug substances or finished products as a result of manufacturing, storage, or handling.
Types of Impurities
Process-related → unreacted starting materials, by-products, catalysts.
Degradation-related → formed through heat, oxidation, hydrolysis, or light.
Residual solvents → examples include methanol and dichloromethane.
Enantiomeric impurities → incorrect stereoisomers in chiral drugs.
According to ICH Q3A/Q3B, any impurity at or above 0.1% must be identified, with stricter thresholds for genotoxic impurities.
Identifying Impurities Using HPLC
Detection
The main API peak appears alongside smaller impurity peaks.
Characterization
Compare retention times with reference standards.
Use PDA detectors to check peak purity.
Confirm identity by co-injection with standards.
Structural Identification
LC-MS provides molecular weight and fragmentation patterns.
LC-NMR or LC-FTIR supplies structural fingerprints.
Together, these techniques give the complete chemical identity.
Examples of Well-Known Impurities
Nitrosamines (e.g., NDMA, NDEA)
Discovered in valsartan, ranitidine, and metformin between 2018–2019.
Classified as probable human carcinogens.
Regulatory limits are extremely low (nanogram levels).
Identified using HPLC-MS/MS.
p-Aminophenol (Paracetamol)
A toxic degradation product affecting liver and kidney.
Strictly limited to ≤0.1%.
Formaldehyde / Acetaldehyde
Residual solvent-related impurities found in excipients.
Detected using derivatization followed by HPLC.
Epimer impurities in chiral drugs
Wrong stereoisomers can be inactive or harmful.
The thalidomide case highlighted the risks.
Controlled and identified using chiral HPLC columns.
Why Impurity Profiling is Essential
Patient safety → prevents toxic exposure.
Regulatory compliance → required by ICH and FDA.
Process understanding → reveals weaknesses in synthesis or storage.
Shelf-life assurance → guarantees product safety until expiry.
Key Takeaway
Finding an extra peak in an HPLC chromatogram is only the first step. Comprehensive quality control requires:
Detection – observing the impurity peak.
Identification – determining chemical structure with advanced techniques.
Control – ensuring levels remain below regulatory limits.
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Serial dilution is a fundamental laboratory technique used to reduce concentration step by step. It ensures accurate analysis of pharmaceutical compounds, supports method validation, and provides consistency across regulatory standards.
What is Serial Dilution?
Serial dilution involves repeatedly diluting a solution by a fixed factor (commonly 1:10 or 1:100) using a solvent. This creates a series of decreasing concentrations and is widely applied in:
Analytical method validation
Microbiological assays
Pharmaceutical quality control
Why is it Essential?
Precision: Enables preparation of reliable low-concentration standards for calibration and testing.
Consistency: Provides uniform preparation to support inter-laboratory reproducibility and compliance with regulatory requirements.
Versatility: Useful for preparing standards, quantifying active ingredients, and studying dose-response relationships.
Step-by-Step Guide
Prepare a stock solution by accurately weighing or measuring the compound and dissolving it in a suitable solvent.
Dilute sequentially by transferring a fixed volume of the stock into successive containers with solvent, mixing thoroughly at each step.
Document concentrations carefully to ensure traceability and accuracy.
Serial dilution is not just a routine laboratory practice—it is a strategic process that underpins precision, reliability, and the success of pharmaceutical research and regulatory compliance.
#PharmaceuticalScience #MethodValidation #SerialDilution #LabTechniques #RegulatoryAffairs #DrugDevelopment #LinkedInLearning #Labtip #analytical #HPLC #RP_HPLC #FTIR #Pharmaceutical_sciences #drugs
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