# MIX-MB(X) — Xenobiotics

## Xenobiotics Component Standard (X)

This document identifies the Minimum Information (MI) required to report xenobiotics involved in microbial biotransformation studies, ensuring comprehensive documentation of chemical substrates and their transformation products.

**Author:** Mahnoor Zulfiqar **Part of:** MIX-MB Standard\_main\
**Compatible with:**

* MIX-MB(M)
* MIX-MB(B)

**Alignment:** BioSchemas, ChEMBL, ChEBI, ChemONT, MSI Standards, FAIR principles

***

***

## 1. Overview

MIX-MB(X) establishes minimum information standards for reporting microbial biotransformation of xenobiotics, ensuring data is:

* **Findable:** Properly annotated with identifiers and metadata
* **Accessible:** Standardized formats for submission to public repositories
* **Interoperable:** Uses community ontologies and controlled vocabularies
* **Reusable:** Provenance and methodology information

### 1.4 Identifiers and Cross-Referencing

**This is the first practical step: name your compounds properly before filling in any other field.**

#### Compound Index (CIDX)

Each compound in a MIX-MB submission must be assigned a **CIDX** — a study-local identifier that ties together `COMPOUND_RECORD.tsv`, `COMPOUND_CTAB.sdf`, and `ACTIVITY.tsv`.

**Minting rules:**

* Format: `CIDX` followed by a zero-padded integer, e.g. `CIDX0001`, `CIDX0002`
* Assign sequentially within a study; CIDXs do not need to be globally unique
* Assign one CIDX per distinct chemical structure (identical canonical SMILES = same CIDX)
* For biotransformation products that are detected but not fully characterised, use the `UNKNOWN_` or `PUTATIVE_` prefix scheme (see below)

#### External Identifier Priority Order

When referencing a compound in an external database, use the following priority order:

| Priority | Identifier      | Field                               | Example                       |
| -------- | --------------- | ----------------------------------- | ----------------------------- |
| 1        | **InChIKey**    | `inChIKey`                          | `HEFNNWSXXWATRW-UHFFFAOYSA-N` |
| 2        | **ChEMBL ID**   | `identifier`                        | `CHEMBL1201246`               |
| 3        | **PubChem CID** | `identifier`                        | `CID:4583`                    |
| 4        | **ChEBI ID**    | `identifier`                        | `CHEBI:3686`                  |
| 5        | **CAS Number**  | `additionalProperty` → `cas_number` | `42399-41-7`                  |

The **InChIKey** is the canonical structure identifier in MIX-MB and is **mandatory** for all known compounds. It identifies the chemical structure independently of naming conventions or database-specific numbering.

#### Minting Scheme for Unknown Compounds

For biotransformation products that cannot be fully identified at the time of submission, use the following naming convention:

| Situation                                         | CIDX format           | MSI Level | Example               |
| ------------------------------------------------- | --------------------- | --------- | --------------------- |
| Confirmed structure                               | `CIDX[nnnn]`          | Level 1–2 | `CIDX0003`            |
| Putatively characterised (structural class known) | `PUTATIVE_[RIDX]_[n]` | Level 3   | `PUTATIVE_GutMeta_P1` |
| Unknown (mass only, no formula or structure)      | `UNKNOWN_[RIDX]_[n]`  | Level 4–5 | `UNKNOWN_GutMeta_M3`  |

* `[RIDX]` is the reference index for the study (e.g. `GutMeta`)
* `[n]` is a sequential integer within the study
* Once an unknown compound is formally identified, replace its `UNKNOWN_` CIDX with a standard CIDX and update all linked `ACTIVITY.tsv` rows accordingly

***

## 2. Bioschemas

MIX-MB(X) integrates already established standards such as BioSchema profile [Bioschemas ChemicalSubstance](https://bioschemas.org/types/ChemicalSubstance/) and [Bioschemas MolecularEntity](https://bioschemas.org/profiles/MolecularEntity/). BioSchemas fulfils Findability aspect of FAIR.

***NOTE:*** ChemicalSubstance is not inclusive of all fields present in the Bioschemas ChemicalSubstance Profile, some are specific to this use case.

### 2.1 Xenobiotics -- ChemicalSubstance Profile with Concentration

The parent compound or xenobiotic used within the research follows [Bioschemas ChemicalSubstance](https://bioschemas.org/types/ChemicalSubstance/) with concentration and sample information captured as properties:

**Required Properties:**

* `@context`: <https://schema.org/>
* `@type`: ChemicalSubstance
* `@id`: Globally unique IRI identifying the compound (e.g., canonical database URL such as `https://www.ebi.ac.uk/chembl/compound_report_card/CHEMBL1201246/`)
* `dct:conformsTo`: <https://bioschemas.org/profiles/ChemicalSubstance/0.4-RELEASE>
* `identifier`: Compound identifier as Text, PropertyValue, or URL (e.g., ChEMBL ID, PubChem CID, InChI Key)
* `name`: Chemical name of the compound (Text)
* `url`: URL of the compound's entry in a reference database (e.g., ChEMBL, PubChem, ChEBI)
* `inChIKey`: Standard InChI Key
* `identifier`: Compound identifier (ChEMBL ID, PubChem CID, InChI Key)
* `smiles`: Canonical SMILES string *(MIX-MB extension)*
* `chemicalComposition`: Chemical formula (e.g: Molecular formula in this context)
* `molecularWeight`: Molecular weight with unit \[Units should be included in the form ‘ ‘, for example ‘12 amu’.]

**Recommended Properties:**

* `alternateName`: An alias for the item (Text, MANY) — e.g., trade names, synonyms, preferred compound name
* `iupacName`: IUPAC systematic name *(from MolecularEntity profile, not ChemicalSubstance)*
* `monoisotopicMolecularWeight`: Exact monoisotopic mass *(from MolecularEntity profile, not ChemicalSubstance)*
* `chemicalRole`: A role played by the molecular entity within a chemical context (DefinedTerm, MANY) — use ChEBI terms e.g., xenobiotic, drug, metabolite
* `associatedDisease`: A disease associated with this BioChemEntity (MedicalCondition or URL, MANY)
* `bioChemInteraction`: A BioChemEntity this compound interacts with (BioChemEntity, MANY) — e.g., drug targets, enzymes, receptors
* `bioChemSimilarity`: A similar BioChemEntity, e.g., obtained by fingerprint similarity algorithms (BioChemEntity, MANY)
* `biologicalRole`: A role played by the BioChemEntity within a biological context (DefinedTerm, MANY) — use ChEBI terms e.g., enzyme inhibitor, antimicrobial agent
* `description`: A free-text description of the item (Text, ONE)
* `disambiguatingDescription`: A short description used to disambiguate from other similar items (Text, ONE)
* `image`: A structural image of the compound (ImageObject or URL, MANY) — e.g., 2D/3D depiction
* `potentialUse`: Intended use of the BioChemEntity by humans (DefinedTerm, MANY) — e.g., pharmaceutical, pesticide, food additive
* `sameAs`: URL of a reference web page that unambiguously indicates the item's identity in another database (URL, MANY)

**Sample Properties (using `additionalProperty`) not available in the ChemicalSubstance Profile:**

* `cas_number`: CAS Registry Number of the compound
* `vendor`: Supplier/vendor who provided the compound (e.g., Sigma-Aldrich, TCI)
* `purity`: Purity percentage and measurement method (e.g., ≥98%, HPLC)
* `solubility`: Solubility information (solvent, concentration limit)
* `stock_concentration`: Stock solution concentration with unit and solvent
* `stock_solvent`: Solvent used to prepare the stock solution (e.g., DMSO, ethanol, water)
* `storage_conditions`: Storage conditions and stability notes (e.g., -20°C, protect from light)
* `local_synonym`: Local study-specific synonym or identifier used in the manuscript (e.g., compound 23)

**Analytical Detection Properties (using `additionalProperty`):**

* `mz`: Measured m/z value of the compound or eluted form
* `column_separation`: Separation technique used (e.g., LC, GC)
* `retention_time`: Retention time recorded during separation
* `retention_time_unit`: Unit for retention time (sec, min, hr)
* `eluted_compound`: Whether the eluted compound differs from the original (e.g., yes for salts where MS detects the active form)
* `eluted_compound_smiles`: SMILES of the eluted compound if different from the original

***NOTE:*** This profile goes to the `COMPOUNDS_RECORD.tsv`, `COMPOUNDS_CTAB.sdf`, `ASSAY.tsv` and `ASSAY_PARAM.tsv` files for ChEMBL submission.

### 2.2 Products and Metabolites with MSI Levels

For biotransformation products characterised with mass spectrometry (MS), use the same **MolecularEntity profile** with **MSI identification confidence levels** to indicate the degree of characterization. This profile is optional and is only important in case if a xenobiotics product is measured and detected.

**MSI Level Guidelines:**

Levels 1–5 follow the updated standard [Metabolomics Standards Initiative (MSI)](https://metabolomicssociety.org/resources/metabolomics-standards-initiative) scheme.

| Level       | Description              | Confidence | Required Information                               | Example                                                                         |
| ----------- | ------------------------ | ---------- | -------------------------------------------------- | ------------------------------------------------------------------------------- |
| **Level 1** | Identified               | Highest    | Authenticated with authentic reference standard    | HMDB ID, exact retention time match, MS² match to standard                      |
| **Level 2** | Putatively annotated     | High       | Library or database match with MS² similarity      | MS² similarity >0.8, retention index agreement, spectral match to database      |
| **Level 3** | Putatively characterized | Medium     | Chemical class identified, partial structure known | Molecular formula confirmed, functional group identified, structure class known |
| **Level 4** | Unknown                  | Low        | Minimal structural information                     | Exact mass only, no formula or structural information                           |
| **Level 5** | Unidentified             | Unknown    | Spectral data only, no structural hypothesis       | Mass peak at m/z 254.1154, no formula or structure information                  |

**Required Properties (for all levels):**

* `@context`: <https://schema.org/>
* `@type`: MolecularEntity
* `@id`: Globally unique IRI identifying the product (e.g., canonical database URL, or a study-local URI for unknowns such as `#UNKNOWN_M3`)
* `dct:conformsTo`: <https://bioschemas.org/profiles/MolecularEntity/0.7-DRAFT>
* `molecularWeight`: Molecular weight with unit (QuantitativeValue or Text, ONE) — use UO unit codes (see Section 3.3); *Recommended in profile, Required for MIX-MB*
* `identifier`: Product identifier as DefinedTerm, PropertyValue, Text, or URL (use `UNKNOWN_` prefix for unknowns)
* `isPartOfBioChemEntity`: Reference to the parent xenobiotic (BioChemEntity, MANY)
* `chemicalRole`: Must include "metabolite" and/or "biotransformation product" (DefinedTerm, MANY) — use ChEBI terms
* `additionalProperty`: Must include MSI identification level (PropertyValue)

**Recommended Properties (if known):**

* `inChIKey`: Hashed InChI identifier (Text, ONE) — *Recommended in profile*
* `iupacName`: IUPAC systematic name (Text, ONE) — *Recommended in profile*
* `molecularFormula`: Molecular formula (Text, ONE) — *Recommended in profile*
* `molecularWeight`: Molecular weight with unit (QuantitativeValue or Text, ONE) — *Recommended in profile, Required for MIX-MB*
* `sameAs`: Reference URLs unambiguously indicating the product’s identity in other databases (URL, MANY) — *Recommended in profile*
* `alternateName`: Product name or systematic designation (Text, MANY)
* `associatedDisease`: A disease associated with this entity (MedicalCondition, PropertyValue, or URL, MANY)
* `bioChemInteraction`: A BioChemEntity this product interacts with (BioChemEntity, MANY)
* `bioChemSimilarity`: A similar molecular entity, e.g., obtained by fingerprint similarity algorithms (BioChemEntity, MANY)
* `biologicalRole`: A role played by the entity in a biological context (DefinedTerm, MANY) — use ChEBI terms
* `chemicalRole`: A role played by the molecular entity within a chemical context (DefinedTerm, MANY) — *Optional in profile, Required for MIX-MB*
* `description`: A free-text description of the product (Text, ONE)
* `disambiguatingDescription`: A short description to distinguish from similar items (Text, ONE)
* `hasBioChemEntityPart`: A BioChemEntity that (in some sense) has this entity as a part (BioChemEntity, MANY)
* `hasMolecularFunction`: Molecular function performed by this entity (DefinedTerm, PropertyValue, Text, or URL, MANY) — use GO terms
* `hasRepresentation`: A common representation of this entity such as a chemical structure or structural classification (DefinedTerm, PropertyValue, Text, or URL, MANY) — e.g., SMILES, InChI, or ChemOnt/ClassyFire class (see Section 3.2)
* `image`: A structural image of the compound (ImageObject or URL, MANY)
* `isEncodedByBioChemEntity`: A gene or BioChemEntity encoding this entity (Gene, MANY)
* `isInvolvedInBiologicalProcess`: Biological process this entity is involved in (DefinedTerm, PropertyValue, Text, or URL, MANY) — use GO terms (see Section 3.4)
* `isLocatedInSubcellularLocation`: Subcellular location where this entity is located (DefinedTerm, PropertyValue, Text, or URL, MANY)
* `isPartOfBioChemEntity`: The parent BioChemEntity this entity is a part of (BioChemEntity, MANY) — *Optional in profile, Required for MIX-MB*
* `monoisotopicMolecularWeight`: Exact monoisotopic mass (QuantitativeValue or Text, ONE)
* `potentialUse`: Intended use of the entity by humans (DefinedTerm, MANY)
* `subjectOf`: A CreativeWork or Event about this entity (CreativeWork or Event, MANY) — e.g., publication, dataset
* `taxonomicRange`: Taxonomic grouping of the organism that expresses or relates to this entity (DefinedTerm, Taxon, Text, or URL, MANY)

**Analytical Detection Properties (using `additionalProperty`):**

*Required for all MSI levels:*

* `MSI_level`: MSI identification confidence level assigned to this product (Level 1–5; see MSI Level Guidelines above)
* `mz`: Measured m/z value of the detected ion
* `adduct_type`: Ion adduct form detected (e.g., \[M+H]+, \[M-H]-, \[M+Na]+, \[M+NH4]+)
* `ionization_mode`: Ionization mode used (e.g., ESI positive, ESI negative, APCI)
* `mass_accuracy_ppm`: Mass measurement accuracy relative to theoretical mass (parts per million)
* `retention_time`: Retention time recorded during chromatographic separation
* `retention_time_unit`: Unit for retention time (sec, min, hr)
* `signal_to_noise`: Signal-to-noise ratio of the detected peak
* `detection_frequency`: Number of replicates in which the compound was detected (e.g., 3/3)
* `transformation_type`: Type of biotransformation from the parent compound (use controlled vocabulary from Section 3.5)
* `mass_difference_from_parent`: Exact mass difference from the parent xenobiotic (e.g., +15.9949 Da for hydroxylation)

*Level 1 (Identified) — additional required:*

* `reference_standard`: Authentic reference standard used for confirmation (supplier, catalog number)
* `retention_time_match`: Retention time difference compared to the authentic reference standard (with unit)
* `ms2_similarity_score`: MS/MS spectral similarity score vs the reference standard spectrum (0–1)

*Level 2 (Putatively annotated) — additional required:*

* `ms2_similarity_score`: MS/MS spectral similarity score vs matched library spectrum (e.g., cosine similarity ≥ 0.7)
* `spectral_library`: Spectral library used for matching (e.g., GNPS, MassBank, HMDB, mzCloud)
* `library_match_id`: Identifier of the matched library spectrum (e.g., GNPS accession, MassBank ID)
* `retention_index_match`: Agreement between experimental and literature retention index (if applicable)

*Level 3 (Putatively characterized) — additional required:*

* `structural_hypothesis`: Proposed structural class or transformation type (e.g., double hydroxylation at aliphatic chain)
* `fragment_pattern`: Key fragment ions supporting the structural assignment (e.g., m/z 177, 159, 137)
* `chemont_class`: ClassyFire/ChemOnt structural classification of the putative compound (use in `hasRepresentation`; maps to template `Metabolite_annotation` column) — e.g., "Phenylpropanoic acids", "Dihydroxylated phenylpropionic acid"; see Section 3.2

*Level 4/5 (Unknown/Unidentified) — additional required:*

* `ms2_available`: Whether an MS/MS spectrum was acquired for this signal (yes/no)
* `possible_molecular_formula`: Proposed molecular formula derived from accurate mass measurement (± ppm tolerance)
* `detection_consistency`: Number of replicates with a confirmed signal (e.g., 2 out of 3)

***NOTE:*** This profile goes to the `ASSAY.tsv` and `ACTIVITY.tsv` files for ChEMBL submission.

### 2.3 Concentration Documentation

Concentration is documented using `additionalProperty` (PropertyValue) for both substrates (Section 2.1) and biotransformation products (Section 2.2). All five properties below apply to both, though `initial_concentration` and `stock_concentration` are most relevant to substrates, while `final_concentration` and `concentration_change` are most relevant to products.

| Property                | Description                                                                                                                           | Recommended Units    | Example                           |
| ----------------------- | ------------------------------------------------------------------------------------------------------------------------------------- | -------------------- | --------------------------------- |
| `stock_concentration`   | Concentration of the compound in the prepared stock solution, before dilution into the assay medium                                   | mmol/L, mg/mL, µg/µL | 100 mmol/L in DMSO                |
| `initial_concentration` | Starting concentration of the compound in the assay at time zero (t=0), after dilution from stock                                     | µM, µg/mL, mmol/L    | 50 µM                             |
| `final_concentration`   | Measured concentration of the compound (substrate remaining or product formed) at the experimental endpoint or a defined timepoint    | µM, µg/mL, nmol/L    | 2.5 µM at 24 h                    |
| `concentration_change`  | Absolute or relative change in concentration compared to the initial value — use to report substrate degradation or product formation | µM, %, fold-change   | 95% degradation; +45.2 µM product |
| `detection_limit`       | Lower limit of quantitation (LLOQ) for the analytical method used; signals below this value should not be reported as quantified      | nmol/L, µg/mL, ppm   | 10 nM                             |

***

## 3. Ontologies

### 3.1 ChEBI (Chemical Entities of Biological Interest)

**Required ChEBI Classifications:**

* **Chemical Role** (`chemicalRole`): Intrinsic chemical role of the compound (e.g., CHEBI:50906 'xenobiotic', CHEBI:25212 'metabolite') — for biotransformation products (§2.2), must include CHEBI:25212 'metabolite'
* **Biological Role** (`biologicalRole`): How the compound functions in a biological context (e.g., CHEBI:38439 'calcium channel blocker', CHEBI:33281 'antimicrobial agent', CHEBI:35222 'inhibitor')
* **Application** (`potentialUse`): Intended therapeutic or practical use (e.g., CHEBI:35610 'drug', CHEBI:49295 'pharmaceutical', CHEBI:35842 'analgesic')

**Example Annotations:**

```
Diltiazem (substrate):
  Chemical Role:   CHEBI:3686 (diltiazem), CHEBI:50906 (xenobiotic)
  Biological Role: CHEBI:38439 (calcium channel blocker)
  Application:     CHEBI:35474 (antihypertensive agent), CHEBI:35498 (antiarrhythmic drug)

N-desmethyldiltiazem (biotransformation product):
  Chemical Role:   CHEBI:25212 (metabolite)
  Biological Role: CHEBI:38439 (calcium channel blocker)
```

### 3.2 ChemOnt (Chemical Ontology)

Use [ClassyFire Chemical Ontology](http://classyfire.wishartlab.com/) for structural classification:

**Required Levels:**

1. **Kingdom:** Highest level (e.g., Organic compounds, Inorganic compounds)
2. **Superclass:** Major structural category (e.g., Organoheterocyclic compounds)
3. **Class:** Structural class (e.g., Benzene and substituted derivatives)
4. **Subclass:** Detailed structural features (optional)

**Example:**

```
Diltiazem:
  Kingdom: Organic compounds
  Superclass: Organoheterocyclic compounds
  Class: Benzothiazepines
  Subclass: 1,5-Benzothiazepines
  Direct Parent: 1,5-Benzothiazepines
```

### 3.3 Unit Ontology (UO)

Use [Unit Ontology](https://bioportal.bioontology.org/ontologies/UO) for standardized unit codes in quantitative measurements. UO ensures interoperability of measurement data across databases and computational tools.

**Required Unit Codes:**

* **Mass:** `GM` (gram), `DA` (dalton), `MGM` (milligram), `UGM` (microgram)
* **Volume:** `ML` (milliliter), `UL` (microliter), `NL` (nanoliter)
* **Concentration:** `MMO` (millimolar), `UMO` (micromolar), `NMO` (nanomolar), `PMO` (picomolar)
* **Time:** `MIN` (minute), `HOUR` (hour), `DAY` (day), `SEC` (second)
* **Temperature:** `CEL` (degree Celsius), `DEK` (kelvin)
* **pH:** Dimensionless (no unit code)
* **Percentage:** `%` (percent)

**Example - Concentration and Quantitation Units:**

```yaml
concentrations:
  stock_concentration:
    value: 100
    unitCode: MMO
    description: "millimolar (mmol/L)"
  
  metabolite_concentration:
    value: 45.2
    unitCode: UMO
    description: "micromolar (µM)"
  
  exact_mass:
    value: 222.1256
    unitCode: DA
    description: "dalton (unified atomic mass unit)"
  
  molecular_weight:
    value: 206.28
    unitCode: GM
    description: "gram per mole (g/mol)"
  
  retention_time:
    value: 4.32
    unitCode: MIN
    description: "minute"
  
  substrate_degradation:
    value: 95
    unitCode: "%"
    description: "percent"
```

### 3.4 Gene Ontology (GO) - Biotransformation Processes

Use [Gene Ontology](http://geneontology.org/) to classify biotransformation processes and enzymatic reactions. GO provides standardized terms for biological processes that enable semantic linking with genomic and proteomic data.

**Phase I - Oxidation/Reduction/Hydrolysis Terms:** Phase I refers to the xenobiotics modification via common enzymatics reactions such as oxidation, reduction and hydrolysis

* GO:0018126 (hydroxylation)
* GO:0055114 (oxidation-reduction)
* GO:0004435 (phosphodiesterase activity)
* GO:0008941 (glucuronidase activity - Phase II)

**Phase II - Conjugation Terms:** Phase II refers to the further modification of the xenobiotics metabolite from phase I, via conjugation of methyl, glycosul, alkyl or sulfide groups.

* GO:0016757 (transferase activity, transferring glycosyl groups)
* GO:0008194 (UDP-glycosyltransferase activity)
* GO:0016620 (transferase activity, transferring alkyl or aryl groups)
* GO:0008146 (sulfotransferase activity)

**Common Biotransformation GO Terms Reference:**

| Transformation  | GO Term                              | GO ID      |
| --------------- | ------------------------------------ | ---------- |
| Hydroxylation   | Hydroxylation                        | GO:0018126 |
| Glucuronidation | UDP-glucuronosyltransferase activity | GO:0008194 |
| Sulfation       | Sulfotransferase activity            | GO:0008146 |
| Acetylation     | Acetyltransferase activity           | GO:0016408 |
| Oxidation       | Oxidation-reduction process          | GO:0055114 |
| Reduction       | Oxidation-reduction process          | GO:0055114 |
| Hydrolysis      | Hydrolase activity                   | GO:0016787 |
| Deamination     | Protein deamination                  | GO:0018133 |
| Conjugation     | Transferase activity                 | GO:0016757 |
| Methylation     | Methyltransferase activity           | GO:0008168 |

### 3.5 MIX-MB Controlled Vocabularies

These terms must be used exactly as written to ensure consistency across submissions.

**Transformation types** (use in `additionalProperty` → `transformation_type`, and in template `Reaction_type` column):

These terms map directly to GO terms (see Section 3.4). Use the MIX-MB term as the `value` and the GO URI as `valueReference` when expressing in JSON-LD.

| MIX-MB Term       | Description                          | GO ID      | GO URI                                        |
| ----------------- | ------------------------------------ | ---------- | --------------------------------------------- |
| `hydroxylation`   | Addition of -OH group                | GO:0018126 | <http://purl.obolibrary.org/obo/GO\\_0018126> |
| `oxidation`       | Oxidation reaction                   | GO:0055114 | <http://purl.obolibrary.org/obo/GO\\_0055114> |
| `reduction`       | Reduction reaction                   | GO:0055114 | <http://purl.obolibrary.org/obo/GO\\_0055114> |
| `hydrolysis`      | Hydrolytic cleavage                  | GO:0016787 | <http://purl.obolibrary.org/obo/GO\\_0016787> |
| `decarboxylation` | Loss of CO₂                          | GO:0017001 | <http://purl.obolibrary.org/obo/GO\\_0017001> |
| `deamination`     | Loss of amino group                  | GO:0018133 | <http://purl.obolibrary.org/obo/GO\\_0018133> |
| `conjugation`     | Addition of a larger molecular group | GO:0016757 | <http://purl.obolibrary.org/obo/GO\\_0016757> |
| `demethylation`   | Loss of methyl group                 | GO:0032259 | <http://purl.obolibrary.org/obo/GO\\_0032259> |
| `acetylation`     | Addition of acetyl group             | GO:0016408 | <http://purl.obolibrary.org/obo/GO\\_0016408> |
| `glucuronidation` | Addition of glucuronic acid          | GO:0008194 | <http://purl.obolibrary.org/obo/GO\\_0008194> |
| `sulfation`       | Addition of sulfate group            | GO:0008146 | <http://purl.obolibrary.org/obo/GO\\_0008146> |

**Activity ACTION\_TYPE** (use in ACTIVITY.tsv):

| Term                | Meaning                           |
| ------------------- | --------------------------------- |
| `Biotransformation` | General transformation event      |
| `Substrate`         | Compound consumed in the reaction |
| `Product`           | Compound produced by the reaction |
| `No Activity`       | No biotransformation detected     |

***

### 4. Data Validation Rules

Before submitting data, verify that all compound records pass the following checks. The pipeline (`generate_compound_files.py`) performs these checks automatically; they also apply to manually prepared submissions.

#### 4.1 Structural Identifiers

| Rule                        | Check                                                                      |
| --------------------------- | -------------------------------------------------------------------------- |
| SMILES is present           | Every compound must have a non-empty canonical SMILES string               |
| SMILES is valid             | SMILES must parse without errors (use RDKit or OpenBabel to verify)        |
| InChIKey is present         | Mandatory for all known compounds (MSI Level 1–2)                          |
| InChIKey format             | Must match the pattern `[A-Z]{14}-[A-Z]{10}-[A-Z]` (27 characters)         |
| SMILES–InChIKey consistency | InChIKey must be derivable from the provided SMILES (no manual mismatches) |

#### 4.2 Compound Index (CIDX)

| Rule            | Check                                                                                                |
| --------------- | ---------------------------------------------------------------------------------------------------- |
| CIDX is present | Every row in `COMPOUND_RECORD.tsv` must have a CIDX                                                  |
| CIDX format     | Must follow `CIDX` + zero-padded integer, e.g. `CIDX0001`                                            |
| CIDX uniqueness | Each CIDX must be unique within the study                                                            |
| Unknown prefix  | Uncharacterised compounds must use `UNKNOWN_[RIDX]_[n]` or `PUTATIVE_[RIDX]_[n]` — never a bare CIDX |

#### 4.3 Required Fields

The following fields must be non-empty in `COMPOUND_RECORD.tsv` for every compound:

* `CIDX`, `RIDX`, `COMPOUND_NAME`

The following fields must be non-empty in `COMPOUND_CTAB.sdf` for every compound:

* Canonical SMILES, InChI, InChIKey, CIDX (as SDF property tag)

#### 4.4 Concentration and Units

| Rule                        | Check                                                                                        |
| --------------------------- | -------------------------------------------------------------------------------------------- |
| Unit codes are standardised | All concentration values must use UO unit codes (see Section 3.3): `MMO`, `UMO`, `NMO`, etc. |
| Numeric values are positive | Concentration, molecular weight, and retention time values must be > 0                       |
| Detection limit is provided | If quantitative data are reported, `detection_limit` should be included                      |

#### 4.5 MSI Level Consistency

| MSI Level | Required fields                                                                |
| --------- | ------------------------------------------------------------------------------ |
| Level 1   | InChIKey, `reference_standard`, `ms2_similarity_score`, `retention_time_match` |
| Level 2   | InChIKey, `ms2_similarity_score`, `spectral_library`, `library_match_id`       |
| Level 3   | `structural_hypothesis`, `fragment_pattern`, `chemont_class`                   |
| Level 4/5 | `mz`, `ms2_available`, `detection_consistency`                                 |

***

### 5. Data Quality Tiers

MIX-MB(X) defines three compliance tiers for xenobiotic compound data. Higher tiers enable broader reuse, database cross-linking, and publication in FAIR-compliant repositories.

#### Tier 1 — Gold (Publication-Ready)

Meets all mandatory and recommended fields. Suitable for ChEMBL submission and FAIR data publications.

* InChIKey present and verified against SMILES
* At least one external database ID (ChEMBL, PubChem, or ChEBI)
* `sameAs` links provided for all known compounds
* Full Bioschemas ChemicalSubstance JSON-LD record (Section 2.1)
* Purity, vendor, and stock concentration documented
* Concentration data complete: initial, final, and detection limit
* For detected products: MSI Level 1 or 2 with full analytical evidence

#### Tier 2 — Silver (Research-Grade)

Meets all mandatory fields with partial recommended fields. Suitable for internal data sharing and preprint deposition.

* InChIKey present
* At least one external database ID
* `COMPOUND_NAME`, `SMILES`, `CIDX`, `RIDX` all present
* Initial substrate concentration documented
* For detected products: MSI Level 2 or 3 acceptable

#### Tier 3 — Bronze (Preliminary / Screening)

Meets only mandatory fields. Suitable for large-scale screening data where full characterisation is not yet possible.

* Valid SMILES and `COMPOUND_NAME` present
* `CIDX` and `RIDX` assigned
* Qualitative activity reported (substrate consumed / product detected / no activity)
* Single measurement acceptable; full replication not required at this tier

***

### 6. How to use the Template

The MIX-MB submission template is provided as [Templates/Template.xlsx](https://github.com/zmahnoor14/BioXend/blob/devel/Standards/Templates/Template.xlsx). The **Compounds** sheet covers the MIX-MB(X) standard.

#### Colour coding

| Colour | Meaning                                                       |
| ------ | ------------------------------------------------------------- |
| Green  | Mandatory — must be filled for a valid submission             |
| Blue   | Recommended — strongly encouraged; required for Tier 1 (Gold) |
| Yellow | Optional — fill if available                                  |

#### Step-by-step

1. **Open** `Template.xlsx` and go to the **Compounds** sheet.
2. **Add one row per compound.** If the same compound appears in multiple assays, it still gets only one row here — the link to assays is made in the Activity sheet.
3. **Fill in the CIDX** — assign sequentially (`CIDX0001`, `CIDX0002`, …). Use `UNKNOWN_` or `PUTATIVE_` prefixes for uncharacterised compounds (see Section 1.4).
4. **Fill in the SMILES.** Use canonical SMILES. If you have a structure drawn in ChemDraw or Marvin, export as canonical SMILES.
5. **Generate the InChIKey** from the SMILES using any of the following:
   * Python/RDKit: `Chem.MolToInchi(mol)` → `inchi.InchiToInchiKey(inchi)`
   * Online: <https://www.cheminfo.org/Chemistry/Cheminformatics/FormatConverter/index.html>
6. **Look up external database IDs** using the InChIKey:
   * ChEMBL: search by InChIKey at [ebi.ac.uk/chembl](https://www.ebi.ac.uk/chembl/)
   * PubChem: search at [pubchem.ncbi.nlm.nih.gov](https://pubchem.ncbi.nlm.nih.gov/)
7. **Fill in concentration fields** (stock, initial, units) using UO unit codes (Section 3.3).
8. **For biotransformation products,** go to the **Metabolites** sub-section and assign an MSI level before filling in the analytical detection fields.
9. **Validate** your completed sheet against the rules in Section 4 before running the pipeline or submitting.

### 7. Version History

| Version | Date       | Changes |
| ------- | ---------- | ------- |
| 0.1.0   | 2026-02-05 | Draft   |

***

### 8. Data Access and Repository Guidance

#### 8.1 Repository and Standard Documents

The MIX-MB standard documents, submission template, and BioXend pipeline are openly available:

* **Standard documents:** [GitHub — zmahnoor14/BioXend](https://github.com/zmahnoor14/BioXend), under `Standards/`
* **Submission template:** `Standards/Templates/Template.xlsx` (see Section 6)
* **BioXend pipeline:** Nextflow workflow (`main.nf`) in the repository root

#### 8.2 Files Generated by This Standard

MIX-MB(X) compound data feed the chemical structure files required for ChEMBL deposition. The BioXend pipeline (`generate_compound_files.py`) produces:

| Output File           | Content                                                        |
| --------------------- | -------------------------------------------------------------- |
| `COMPOUND_RECORD.tsv` | Compound metadata indexed by CIDX                              |
| `COMPOUND_CTAB.sdf`   | 2D chemical structures with embedded CIDX, InChI, and InChIKey |
| `chembl_mapping.tsv`  | CIDX–ChEMBL ID cross-reference for known compounds             |

These files are linked to assay and activity records via CIDX (see MIX-MB(B)).

#### 8.3 Submitting to ChEMBL

MIX-MB(X) compound files are deposited to [ChEMBL](https://www.ebi.ac.uk/chembl/) as part of a full MIX-MB submission package. For deposition enquiries, contact **<chembl-help@ebi.ac.uk>** or use the ChEMBL deposition portal.

**Pre-submission checklist for compound data:**

* All compounds pass Section 4 validation rules
* SMILES–InChIKey consistency verified (`generate_compound_files.py` performs this automatically)
* At least one external database ID present (Gold tier) or SMILES + name (Bronze minimum)
* `COMPOUND_CTAB.sdf` contains a valid molblock for every compound

#### 8.4 Supplementary Spectral Data Repositories

Raw spectral data for detected biotransformation products (Section 2.2) should be deposited alongside the ChEMBL submission:

| Repository   | URL                                   | Data type                                    |
| ------------ | ------------------------------------- | -------------------------------------------- |
| MetaboLights | <https://www.ebi.ac.uk/metabolights/> | LC-MS raw data, processed peak tables        |
| GNPS         | <https://gnps.ucsd.edu/>              | MS/MS spectral libraries, molecular networks |
| MassIVE      | <https://massive.ucsd.edu/>           | Raw MS data (alternative to MetaboLights)    |

Record the repository accession in `ACTIVITY_COMMENT` for every row that cites an externally deposited spectrum.

***

### 9. Licence and Reuse

#### 9.1 Standard Documents

The MIX-MB standard documents are released under the **Creative Commons Attribution 4.0 International (CC BY 4.0)** licence (<https://creativecommons.org/licenses/by/4.0/>).

You are free to share, adapt, and build upon this standard for any purpose, provided you give appropriate credit:

> Zulfiqar M. *et al.* MIX-MB: Minimum Information about Xenobiotics-Microbiome Biotransformation (v0.1.0). GitHub: <https://github.com/zmahnoor14/BioXend>

#### 9.2 Pipeline and Code

The BioXend Nextflow pipeline and associated scripts are released under the **MIT Licence**. See `LICENSE` in the repository root for full terms.

#### 9.3 Submitted Data

Data deposited using MIX-MB formats in public repositories are subject to that repository's terms:

| Repository     | Licence                                                          |
| -------------- | ---------------------------------------------------------------- |
| ChEMBL         | CC BY-SA 3.0 (<https://creativecommons.org/licenses/by-sa/3.0/>) |
| MetaboLights   | CC0 1.0 or CC BY 4.0 (depositor's choice at submission)          |
| GNPS / MassIVE | CC0 (public domain)                                              |

When reusing MIX-MB-formatted datasets, cite both the original study (via its RIDX/DOI) and the MIX-MB standard.

***

### 10. Provenance

Provenance records the origin, history, and chain of custody of chemical data reported under MIX-MB(X). Complete provenance supports the **Reusable** principle of FAIR and enables independent verification of compound identities and analytical assignments.

#### 10.1 Study-Level Provenance

Every compound record is anchored to a **Reference record** (RIDX) in `REFERENCE.tsv`, which provides the top-level provenance anchor for the entire submission:

| Field                      | Description                                         |
| -------------------------- | --------------------------------------------------- |
| `RIDX`                     | Study-local identifier linking all submission files |
| `DOI`                      | Digital Object Identifier of the source publication |
| `TITLE`, `AUTHORS`, `YEAR` | Full bibliographic metadata                         |

The RIDX appears in every row of `COMPOUND_RECORD.tsv`, `ASSAY.tsv`, and `ACTIVITY.tsv`, making the publication the root provenance node for all data.

#### 10.2 Compound Identity Provenance

The external identifiers documented in Section 1.4 serve as compound provenance anchors:

| Identifier      | Provenance Source                                                               |
| --------------- | ------------------------------------------------------------------------------- |
| **InChIKey**    | Derived computationally from the SMILES; validated against a reference database |
| **ChEMBL ID**   | Structure matched in ChEMBL via API query (`generate_compound_files.py`)        |
| **PubChem CID** | Structure registered in PubChem                                                 |
| **ChEBI ID**    | Classification from ChEBI Ontology                                              |
| `sameAs` URLs   | Direct links to the authoritative source records                                |

For compounds not found in any external database (novel metabolites, unknowns), the SMILES string and MSI identification level (Section 2.2) together constitute the compound's provenance record.

#### 10.3 Analytical Evidence Provenance

For biotransformation products, the MSI level (Section 2.2) encodes the evidence chain supporting each structural assignment:

| MSI Level | Required Provenance Evidence                                                                                               |
| --------- | -------------------------------------------------------------------------------------------------------------------------- |
| Level 1   | Authentic reference standard; `reference_standard` (supplier, catalog no.), `retention_time_match`, `ms2_similarity_score` |
| Level 2   | Spectral library accession (`library_match_id`), `spectral_library`, match score ≥ 0.7                                     |
| Level 3   | `structural_hypothesis`, `fragment_pattern`, `chemont_class`                                                               |
| Level 4–5 | Observed m/z and `detection_consistency` only                                                                              |

#### 10.4 Pipeline Provenance

When generating submission files with BioXend, record in `REFERENCE.tsv` or `ACTIVITY_COMMENT`:

* **BioXend pipeline version** — from `versions/pipeline.txt`
* **`generate_compound_files.py` version** — from the script header
* **Date of generation** — ISO 8601 format (YYYY-MM-DD)

***

### 11. References

1. ChEMBL Database Guidelines: <https://www.ebi.ac.uk/chembl/>
2. Metabolomics Standards Initiative: <https://metabolomicssociety.org/resources/metabolomics-standards-initiative>
3. Bioschemas Specifications: <https://bioschemas.org/>
4. ChEBI Ontology: <https://www.ebi.ac.uk/chebi/>
5. ClassyFire Chemical Taxonomy: <http://classyfire.wishartlab.com/>
6. Gene Ontology: <http://geneontology.org/>
7. FAIR Principles: <https://www.go-fair.org/fair-principles/>
8. Unit Ontology: <https://bioportal.bioontology.org/ontologies/UO>

***

### 12. Contact and Contributions

For questions, suggestions, or contributions to this standard, please contact:

* **Maintainer:** Mahnoor Zulfiqar
* **Institution:** NFDI4Microbiota
* **Email:** [Contact information](mailto:zmahnoor14@gmail.com)
* **Repository:** \[[GitHub repository URL](https://github.com/zmahnoor14/BioXend)]

This standard is a living document and will be updated based on community feedback and evolving best practices.


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